Process for making chlorocitric acid

ABSTRACT

The present invention relates to chlorocitric acids of the formula ##STR1## and stereoisomers, optical antipodes and pharmaceutically acceptable salts thereof, to methods of preparation thereof, including intermediates involved therein, and to their use as anorectic agents for the treatment of obesity in mammals.

This is a division of application Ser. No. 973,504, filed Dec. 26, 1978,now U.S. Pat. No. 4,312,885.

BACKGROUND OF THE INVENTION

In the continuing war against obesity several derivatives of citricacid, which have been implicated in fatty acid synthesis (see, forexample, A. C. Sullivan et al., Lipids, 9, 121 [1974]), have beenprepared with the hope of developing a medicinal agent effective againstthe disease and devoid of undesirable side effects.

Hydroxycitric acids reduce food intake and body weight gain bysuppressing appetite and inhibiting fatty acid synthesis (A. C. Sullivanet al., Am. J. Clin. Nutr., 30, 767 [1977]). Aminocitric acids areuseful in the treatment of obesity by inhibiting lipogenesis (R. W.Guthrie and R. W. Kierstead, U.S. Pat. No. 3,960,933 [issued June 1,1976]), as are the ester and amide derivatives of hydroxycitric acidα-lactone (R. W. Guthrie and R. W. Kierstead, U.S. Pat. No. 3,993,668[issued Nov. 23, 1976]).

Of the halocitric acids, the fluoro compounds, potent inhibitors ofaconitate hydratase, have been found to be too toxic to be useful in thecontrol of obesity (R. J. Dummell and E. Kun. J. Biol. Chem., 244, 2966(1969).

Chlorocitric acids have been reported in the scientific and patentliterature. D. Pawolleck in Ann., 178, 150 (1875) stated his intentionto prepare "monochlorocitric acid" at page 152 and claimed the formationof a "chlorine containing acid" by the addition of hypochlorous acid toaconitic acid at page 156. By application of Pawolleck's method, C.Martius and R. Mave, Z. Physiol. Chem., 269, 23 (1941) claimed that onlychlorine-free products were obtained. Recently, C. L. Mehltretter, inU.S. Pat. No. 3,536,630 (issued Oct. 27, 1970) reported thatsubstitution of chlorine for hypochlorous acid in Pawolleck's procedureaffords a solution of "disodiumchlorohydroxytricarballylate" in water.Biological properties of the alleged chlorocitric acids were notdisclosed. It has now been found that repetition of the prior methodsfor the addition of hypochlorous acid or chlorine to aconitic acid giveonly non-chlorine containing products.

Iodo- and bromocitric acids have not been described.

DESCRIPTION OF THE INVENTION

The present invention relates to chlorocitric acids of the formula##STR2## and steroisomers, optical antipodes and pharmaceuticallyacceptable salts thereof, to methods of preparation thereof, includingintermediates involved therein, and to their use as anorectic agents inthe treatment of obesity.

More specifically, the present invention relates to(±)-erythro-chlorocitric acid, (±)-threo-chlorocitric acid,(+)-erythro-chlorocitric acid, (-)-erythro-chlorocitric acid,(+)-threo-chlorocitric acid, to methods of preparation thereof involvingthe addition of the elements of hypochlorous acid to trans-aconitic acidto afford (±)-threo-chlorocitric acid, β-lactone, which is hydrolyzed to(±)-threo-chlorocitric acid, the addition of the elements ofhypochlorous acid to cis-aconitic acid to afford(±)-erythro-chlorocitric acid and (±)-threo-chlorocitric acid,β-lactones which are hydrolyzed to (±)threochlorocitric acid, theacid-induced cleavage of (±)-threo- and (±)-erythro-epoxyaconitic acidby alkali metal chloride to (±)-threo-chlorocitric acid and(±)-erythro-chlorocitric acid, respectively, or alternatively, and moreefficiently, the epoxidation of cis-aconitic acid, or the anhydridethereof, in alkaline medium to a partial salt of(±)-erythroepoxyaconitic acid, which is cleaved by alkali metal chloridein the presence of acid to (±)-erythro-chlorocitric acid, theacid-induced cleavage of (+)-threo-epoxyaconitic acid by alkali metalchloride to (-)-threo-chlorocitric acid, the acid-induced cleavage of(-)-threo-epoxyaconitic acid by alkali metal chloride to(+)-threo-chlorocitric acid, the acid-induced cleavage of(-)-erythro-epoxyaconitic acid by alkali metal chloride to(+)-erythro-chlorocitric acid, and the acid-induced cleavage of(+)-erythro-epoxyaconitic acid by alkali metal chloride to(-)-erythro-chlorocitric acid, and to their usefulness as anorecticagents delaying gastric emptying without concomitant metabolic effectson cholesterol or lipid synthesis in the treatment of obesity.

Additionally, the present invention relates to the isolable andcharacterizable mono-salts of (±)-threo-epoxyaconitic acid and itsutility in the synthesis of (±)-threo- and (-)-threo-chlorocitric acidinvolving, respectively, the acid-induced cleavage of the mono-salts of(±)-threo-epoxyaconitic acid to (±)-threo-chlorocitric acid and theneutralization of the mono-salts of (±)-threo-epoxyaconitic acid to(±)-threo-epoxyaconitic acid followed by optical resolution to(+)-threo-epoxyaconitic acid and acid-induced cleavage to(-)-threo-chlorocitric acid.

The present invention also relates to a process for the resolution of(±)-erythrochlorocitric acid employing sequentially(-)-p-nitro-α-methylbenzylamine and (+)-p-nitro-α-methylbenzylamine andto the diastereoisometric salts, (+)-erythro-chlorocitric acid bisR-(-)-α methyl-p-nitrobenzylamine and (-)-erythro-chlorocitric acid bisR-(+)-α-methyl-p-nitrobenzylamine formed thereby.

As used throughout the specification and appended claims, the terms"alkali metal" and "alkaline earth metal" refer to lithium, sodium andpotassium, and calcium, respectively. The term "alkanol" refers to thecompound derived by replacement of a proton of a straight or branchedchain alkane having 1 to 20 carbon atoms by a hydroxyl moiety. Examplesof alkanols include methanol, ethanol, 2-propanol and the like. The term"alkanoic acid" refers to the compound derived by replacement of the twoprotons bound to the carbon atom bearing the hydroxyl moiety of analkanol by a carbonyl function. The term "lower" as applied to theaforementioned groups refers to those groups having 1 to 8 carbon atoms.

The compounds of the present invention, namely, the chlorocitric acidsof formula I and the chlorocitric acid, β-lactones of formula IV, aswell as certain starting materials and intermediates in the preparationthereof, bear two asymmetric centers and thus exist in two relativestereochemical forms: a threo form and an erythro form. Each form, i.e.,the threo form and erythro form, can exist as a racemate and two opticalantipodes, one rotating a beam of polarized light clockwise and theother counterclockwise, the former being designated the (+)-opticalantipode, the latter the (-)-optical isomer and the racemate the(±)-system. In conjunction therewith, the threo-erythro nomenclature asdefined by D. C. Cram and F. A. A. Elhafez, J. Amer. Chem. Soc., 74,5828 (1952) and R. S. Cahn, et al., Experientia, 12, 81 (1956) has beenadopted to facilitate the description of the compound, process andmethods described herein. Thus, for example, the chlorocitric acids ofthe present invention are described as the (±)-erythro-, (+)-erythro-,(-)-erythro-, (±)-threo-, (+)-threo- and (-)-threo-isomers.

While the compounds, processes and methods of the present invention aregenerally depicted for each stereoisomer, a wiggly line ( ) attaching achloro atom or hydroxyl group to the citric acid skeleton has beenemployed to denote all possible orientations of these functions aboutthe asymmetric centers.

(±)-threo-Chlorocitric acid of the formula ##STR3## can be prepared byadding the elements of hypochlorous acid across the carbon-to-carbondouble bond of commercially available trans-aconitic acid of the formula##STR4## to form (±)-threo-chlorocitric acid, β-lactone of the formula##STR5## the β-lactone function of which is hydrolyzed to afford thehydroxyacid II.

The chlorohydrination is conveniently performed by dissolving theaconitic acid III in an aqueous solution of an alkali metal hydroxidesuch as lithium, sodium or potassium hydroxide, preferably sodium orpotassium hydroxide, containing a sufficient amount of alkali metal toform a salt of trans-aconitic acid of the formula ##STR6## wherein M isan alkali metal, cooling the solution to about 0° C. to about 20° C.,preferably to about 5° C., treating the solution with excess chlorine orhypochlorous acid, preferably chlorine, to form a (±)-threo-chlorocitricacid, -lactone salt of the formula ##STR7## wherein M is an alkali metal

and acidifying the salt VI to the acid IV.

Suitable aqueous solvents for the chlorohydrination include water andmixtures of water and lower alkanols such as methanol, ethanol,2-propanol and the like, water and ethers such as dimethoxyethane,tetrahydrofuran and dioxane, and water and polar aprotic solvents suchas dimethylacetamide, dimethylformamide, dimethylsulfoxide andhexamethylphosphoramide.

Among acids suitable for the conversion of the dialkali metal salt of(±)-threo-chlorocitric acid, β-lactone of formula VI to thecorresponding free acid of formula IV may be mentioned mineral acids,for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoricacid and the like, sulfonic acids such as methanesulfonic acid,phenylsulfonic acid, p-toluenesulfonic acid and the like and strongorganic acids such as trifluoroacetic acid, trichloroacetic acid and thelike.

The hydrolysis of the β-lactone function of the diacid of formula IV orthe dialkali metal salt thereof of formula VI is accomplished bysuspending or dissolving the diacid or the disalt in an aqueous solventcontaining an acid such as those employed for the acidification of thedisalt VI to the diacid IV and heating the resulting reaction system toa temperature of about 30° C. to about 80° C. to complete thehydrolysis, which, while relatively slow at room temperature,nevertheless proceeds at a synthetically useful rate at a temperaturewithin this range. A hydrolysis temperature of about 50° C. to 70° C. ispreferred, a hydrolysis temperature of about 70° C. being mostpreferred.

While chlorohydrination of trans-aconitic acid of formula III and thehydrolysis of (±)-threo-chlorocitric acid, β-lactone of formula IV maybe performed stepwise, it is more convenient and efficient to acidifythe dialkali metal salt of formula VI and heat the resulting reactionmixture to complete the hydrolysis of the diacid IV to afford(±)-threo-chlorocitric acid. Thus, upon completion of thechlorohydrination of the aconitic acid of formula III, the reactionmixture is acidified with an acid, preferably a mineral acid, mostpreferably hydrochloric acid, and heated from about 30° C. to about 100°C., preferably from about 50° C. to about 90° C., most preferably atabout 70° C., to complete the conversion of the β-lactone function offormula IV to the hydroxyacid moiety of formula II.

On the basis of the foregoing, one would have anticipated the formationof (±)-erythro-chlorocitric acid of the formula ##STR8## via(±)-erythro-chlorocitric acid, β-lactone of the formula ##STR9## byapplication of the hereinbefore described hypochlorination sequence tocis-aconitic acid of the formula ##STR10## Unexpectedly, the productafter acidification of the reaction is mainly a mixture of(±)-Threo-chlorocitric acid, β-lactone apparently formed byepimerization of the highly strained (±)-erythro-chlorocitric acid,β-lactone, and (±)-threo-chlorocitric acid, (±)-Erythro-chlorocitricacid was formed only in trace amounts.

Alternatively, and more efficiently, (±)-erythro-chlorocitric acid (VII)is prepared in a high yield process involving the cleavage of theepoxide ring of (±)-erythroepoxyaconitic acid of the formula ##STR11##generated in situ by the epoxidation of cis-aconitic acid (IX) or thecorresponding anhydride of the formula ##STR12##

The epoxidation is readily performed utilizing hydrogen peroxide inconjunction with an epoxidation catalyst. A particularly effectiveepoxidation catalyst is tungstic acid or a salt thereof, preferably analkali metal salt, most preferably the sodium salt. While theepoxidation is preferably carried out in water, containing about 0 molarequivalents to about 2.9 molar equivalents of an alkali metal hydroxide,such as lithium, sodium or potassium hydroxide, preferably about 2.5molar equivalents of sodium hydroxide, an organic solvent such as loweralkanol, for example, methanol, ethanol or 2-propanol, or a watersoluble ether such as dimethoxyethane, tetrahydrofuran or dioxane, maybe employed as a diluent. The epoxidation is conveniently performed at areaction temperature of about 0° C. to about 100° C. To minimizeconversion of cis-aconitic acid X or its anhydride XI to thetrans-isomer III, however, it is preferable to perform the epoxidationat a temperature within the range of about 20° C. to about 50° C.

Without isolation, the resulting (±)-erythro-epoxyaconitic acid X or thesalt thereof of the formula ##STR13## wherein M is an alkali metal and Ris hydrogen or M

is acidified and then cleaved by treatment with an alkali metal chloridesuch as lithium, sodium or potassium chloride, preferably sodiumchloride. Suitable acids include mineral acids, for example,hydrochloric acid, sulfuric acid, phosphoric acid and the like, sulfonicacids such as methanesulfonic acid, phenylsulfonic acid,p-toluenesulfonic acid and the like, and strong organic acids such astrifluoroacetic acid, trichloroacetic and the like. Hydrochloric acid ispreferred.

To avoid possible side reactions involving carboxylate ions of theso-formed chloroacid VII, it is desirable to perform the cleavage in thepresence of about one molar equivalent to about 10 molar equivalents ofthe abovementioned acids. Thus, when the epoxidation is carried out inthe absence of an alkali metal hydroxide, it is desirable to employabout one molar equivalent to about 10 molar equivalents of acid, andwhen the epoxidation is accomplished in the presence of about 2.5 molarequivalents of alkali metal hydroxide, it is desirable to utilize about3.5 molar equivalents to about 12.5 molar equivalents of acid. One molarequivalent to 3.5 molar equivalents of acid is preferred.

The epoxide cleavage is usually conducted at a temperature resulting ina convenient reaction rate. While not narrowly critical, the cleavagereaction temperature is normally maintained within the range of about50° C. to about 80° C., preferably at about 70° C., to avoid undesiredside reactions of the chloroacid so-formed.

While the aforedescribed process for the preparation of(±)-erythro-chlorocitric acid (VII) is efficiently performed by cleavingthe (±)-erythro-epoxyaconitic acid X generated in situ, theerythro-epoxy acid X, prepared and isolated by the method disclosed byR. W. Guthrie et al., U.S. Pat. No. 3,969,772, issued June 29, 1976, mayalso be cleaved to the erythro-chloroacid VII as hereinbefore describedfor the in situ process, i.e., by an alkali metal chloride dissolved inan aqueous solvent in the presence of an acid, preferably by excesssodium chloride in the presence of about one mole of hydrochloric acidin water, most preferably at a temperature of about 70° C.

Similarly, (±)-threo-epoxyaconitic acid of the formula ##STR14## whichmay be prepared as described in R. W. Guthrie et al., U.S. Pat. No.3,966,772, issued June 29, 1976, is cleaved by an alkali metal chloridedissolved in an aqueous solvent in the presence of an acid, preferablyby excess sodium chloride dissolved in water in the presence of onemolar equivalent of hydrochloric acid at a temperature within the rangeof about 50° C. to about 80° C., most preferably at a temperature ofabout 70° C., to afford (±)-threo-chlorocitric acid (II).

Likewise, (+)-threo-epoxyaconitic acid of the formula ##STR15## iscleaved to (-)-threo-chlorocitric acid of the formula ##STR16##(-)-threo-epoxyaconitic acid of the formula ##STR17## is cleaved to(+)-threo-chlorocitric acid of the formula ##STR18##(-)-erythro-epoxyaconitic acid of the formula ##STR19## is cleaved to(+)-erythro-chlorocitric acid of the formula ##STR20## and(+)-erythro-epoxyaconitic acid of the formula ##STR21## is cleaved to(-)-erythro-chlorocitric acid of the formula ##STR22## by an alkalimetal chloride dissolved in an aqueous solvent in the presence of anacid, preferably by excess sodium chloride dissolved in water in thepresence of one molar equivalent of hydrochloric acid at a temperaturewithin the range of about 50° C. to about 80° C., most preferably at atemperature of about 70° C.

The synthesis of (+)-threo- and (-)-threo-epoxyaconitic acid by opticalresolution procedures is disclosed in R. W. Guthrie et al. U.S. Pat. No.3,966,772 issued June 29, 1976.

(-)-erythro-Epoxyaconitic acid (XVIII) is prepared from crystalline(-)-threo-hydroxycitric acid of the formula ##STR23## via trimethyl(-)-threo-mesyloxycitrate of the formula ##STR24## the preparation ofwhich is disclosed in R. W. Guthrie et al. U.S. Pat. No. 3,966,772issued June 29, 1976, by a process involving partial hydrolysis of thecarboxylic acid ester groups of the citrate XXIII to a mixture (ca 1:1)of (-)-threo-mesyloxycitric acid of the formula ##STR25## andmethyl(-)-threo-mesyloxycitrate of the formula ##STR26## followed bydisplacement of the mesyloxy group of the di- and tricarboxylic acidXXIV and XXV to form the epoxy function of XVIII and saponification ofthe remaining carboxylic ester group of XXV to complete the synthesis.

The partial hydrolysis is accomplished by treating the mesyloxytricarboxylic acid ester XXIII with a mineral acid such as hydrobromicacid, hydrochloric acid, sulfuric acid, nitric acid and the like in awater miscible solvent. Suitable water miscible solvents include loweralkanols such as methanol, ethanol, 2-propanol and the like, and loweralkanoic acids such as formic, acetic and propionic acids. Theacid-solvent combination of hydrochloric acid-acetic acid is preferred.

To avoid formation of (-)-erythro-hydroxycitric acid, α-lactone of theformula ##STR27## the hydrolysis is preferably performed at a reactiontemperature of about 80° C. for about 24 hours.

The internal displacement of the mesyloxy group of the tricarboxylicacid XXIV and dicarboxylic acid monocarboxylic acid ester XXV withinversion to form the oxirane ring of XVIII and the saponification ofthe carboxylic acid ester group of XXV is conveniently carried outaccording to the method described by R. W. Guthrie et al. in U.S. Pat.No. 3,966,772 issued June 29, 1976.

Appliction of the esterification, mesylation, partial hydrolysis andinternal displacement-saponification sequence to (+)-threo-hydroxycitricacid of the formula ##STR28## affords (+)-erythro-epoxyaconitic acid(XX).

Crystalline (-)-threo-hydroxycitric acid (XXII) is readily prepared fromaminoethyleneammonium (-)-threo-hydroxycitrate of the formula ##STR29##the synthesis of which is reported in U.S. patent application Ser. No.667,317 filed Mar. 16, 1976, now abandoned, by acidifying the saltXXVIII with aqueous trifluoroacetic acid, from which the hydroxy acidXXII crystallizes at a temperature within the range of about 5° C. toabout 10° C. and isolating the product by filtration.

While the optically active chlorocitric acids of the present inventionare more readily prepared by chlorinolysis of the oxirane ring ofoptically active epoxyaconitic acid, as hereinbefore described, thesecompounds may also be prepared from racemic threo- anderythro-chlorocitric acids by resolution methods known in the art. Forexample, by employing (+)-p-nitro-α-methylbenzylamine and(-)-p-nitro-αmethylbenzylamine sequentially as the resolving agents,(±)-erythro-chlorocitric acid (VII) may be resolved into its opticalantipodes, (+)-erythro-chlorocitric acid (XIX) and(-)-erythro-chlorocitric acid (XXI), by separation of thediastereoisomeric salts of the formulas ##STR30## so formed according tothe procedure outlined in U.S. Pat. No. 3,901,915 issued Aug. 26, 1975.

In an additional and highly efficient synthesis of(±)-threo-chlorocitric acid (II), trans-aconitic acid (III) is convertedto its readily isolable and characterizable, highly crystallinemono-alkali metal salt XXX ##STR31## wherein M is alkali metal, which iscleaved by the hereinbefore described methods to the chloroacid II.

While the mono-alkali metal salt of (±)-threo-epoxyaconitic acid isconveniently depicted as structural formula XXX, it is understood thatthe alkali metal may be associated with one of the two other carboxylgroups as shown in formulas XXXI and XXXII. ##STR32## wherein M isalkali metal.

The conversion of trans-aconitic acid (III) to the mono-alkali metalsalt of (±)-threo-epoxyaconitic acid XXX may be accomplished by one ofseveral processes. In the first, trans-aconitic acid (III) istransformed into the di-alkali metal salt of (±)-threo-chlorocitricacid, β-lactone (VI) as herein described. Instead of hydrolyzing theβ-lactone VI directly to the chloroacid II under acidic conditions asherein disclosed, it has been found to be particularly efficacious tofirst hydrolyze the β-lactone function of VI and concomitantly displacethe chloro function to the tri-alkali metal of (±)-threo-epoxyaconiticacid XXXIII ##STR33## wherein M is alkali metal, under alkalineconditions, then partially neutralize the trisalt XXXIII to themono-salt XXX and finally cleave the isolable mono-salt XXX to(±)-threo-chlorocitric acid II by, for example, sodium chloride in thepresence of hydrochloric acid, according to the procedures reportedherein.

The alkali induced hydrolysis-displacement of the dialkali metal salt of(±)-threo-chlorocitric acid, β-lactone (VI) is performed by treating thechlorination reaction mixture with an alkali metal hydroxide such aslithium, sodium or potassium hydroxide, preferably potassium hydroxide,while maintaining the reaction temperature between about 0° C. to about40° C., more preferably to about 0° C. to 25° C.

The partial neutralization of the tri-salt XXXIII is accomplished byadjusting the pH of the hydrolysis-displacement reaction mixture to avalue within the range of about 7.0 to 7.5, preferably to a value ofabout 7.2, cooling the resulting reaction mixture to a temperaturepreferably within the range of above -20° C. to 20° C., more preferablyto a temperature of about 0° C. to 5° C., adding about twomolar-equivalents of acid collecting the precipitate and purifying it byrecrystallization from water or water-alkanol mixtures, such aswater-methanol, water-ethanol and the like. Water is the preferredrecrystallization solvent.

The pH is conveniently adjusted by adding a mineral or organic acid tothe reaction mixture. Among mineral acids, there may be mentionedhydrohalic acids, such as hydrochloric acid, hydrobromine acid, nitricacid, sulfuric acid and perchloric acid and the like. Among organicacids there may be mentioned sulfonic acids such as methanesulfonicacid, benzenesulfonic acid and p-toluenesulfonic acid, trichloroaceticacid and trifluoroacetic acid. Mineral acids are preferred. Hydrohalicacids are more preferred. Hydrochloric acid is most preferred.

In the record process, trans-aconitic acid (III) is chlorohydrinated tothe tri-alkali metal salt of (±)-threo-chlorocitric acid (XXXIV)##STR34## wherein M is alkali metal, which is cyclized under alkalineconditions and partially neutralized under acidic conditions to themono-alkali metal salt of (±)-epoxyaconitic acid (XXXIII).

The chlorohydrination is carried out by treating trans-aconitic acid(III) with an alkali metal hypochlorite, preferably potassiumhypochlorite, preformed by the dissolution of chlorine in an alkalimetal hydroxide solution, preferably, aqueous potassium hydroxide. Whilethe chlorohydrination temperature is not narrowly critical, it ispreferred to perform the reaction at a temperature within the rangeabout -20° C. to about 25° C., more preferably at a temperature withinthe range of about -5° C. to 5° C., most preferably at a temperature ofabout 0° C. About two molar-equivalents of alkali metal hydroxide areinitially employed to from the di-alkali metal salt of trans-aconiticacid (XXXV) ##STR35## which may also be represented as the di-saltsXXXVI and XXXVII. ##STR36## About two additional molar-equivalents ofalkali metal hydroxide are subsequently employed to from sufficientalkali metal hypochlorite for the hypochlorination of di-salt XXXV.

The cyclization of the tri-alkali metal salt of (±)-threo-chlorocitricacid (XXXIV) to the tri-alkali metal salt of (±)-threo-epoxyaconiticacid (XXXIII) is accomplished by treating the chlorohydrination reactionmixture or the tri-alkali metal salt of (±)-threo-chlorocitric acid,dissolved in a suitable solvent, with an alkali metal hydroxide,preferably potassium hydroxide. The cyclization temperature is notnarrowly critical. Nevertheless it is preferred to carry out thereaction at a temperature between about 15° C. to about 60° C., morepreferably at a reaction temperature of about 25° C.

Suitable cyclization solvents include water and mixtures of water andlower alkanols such as methanol, ethanol and the like. Water ispreferred.

The partial neutralization of the tri-alkali metal salt of(±)-trans-epoxyaconitic acid (XXXIII) is effected by treating it, or thereaction mixture in which it is derived, with a mineral or organic acidaccording to the hereinbefore described procedure for the relatedconversion of the di-alkali metal salt of (±)-threo-chlorocitric acid,β-lactam (VI).

In the third process, a variant of the second method, about two-thirdsof a molar-equivalent of trans-aconitic acid is treated with about twomolar-equivalents of an alkali metal hydroxide, preferably potassiumhydroxide, in an appropriate solvent followed by about onemolar-equivalent of preformed alkali metal hypochlorite, preferablypotassium hypochlorite, and about one-third molar-equivalent oftrans-aconitic acid to form (±)-threo-chlorocitric acid (XXXIV).

As appropriate solvent for the third process, there may be mentionedwater and mixtures of water and lower alkanols such as methanol, ethanoland the like. Water is preferred.

While the hypochlorination temperature is not narrowly critical, it ispreferable to perform the reaction at a temperature from about -20° C.to 10° C., more preferably at a temperature of about -10° C. to -5° C.

The (±)-threo-chlorocitric acid (XXXIV), so obtained by this variant, istransformed to the mono-alkali metal salt of (±)-threo-epoxyaconiticacid (XXX) by the methods hereinbefore described in the description ofthe first and second processes.

In the fourth process, (±)-threo-epoxyaconitic acid (XIII) is convertedto the mono-alkali metal salt, preferably the mono-potassium salt, bytreatment of the acid XIII with about one equivalent of an alkali metalhydroxide, preferably potassium hydroxide, in a suitable solvent such aswater and mixtures of water and lower alkanols of the group consistingof methanol, ethanol, 2-propanol and the like. It is preferred to carryout the conversion of the tri-acid XIII to the mono-salt XIII at areaction temperature of about 5° C., although the salt formationtemperature is not critical. The mono-salt XIII so obtained, is isolatedand purified as hereinbefore disclosed for the product of the othervariants.

Alternatively, the reaction mixture in which (±)-threo-epoxyaconiticacid (XIII) is formed may be treated with about one equivalent of analkali metal hydroxide, preferably potassium hydroxide, under theaforementioned conditions.

The mono-alkali metal salts of (±)-threo-epoxyaconitic acid (XXX) angenerally isolated as the monohydrate.

(-)-threo-Chlorocitric acid (XV) may also be prepared from themono-alkali metal salt XXX. In this alternative approach to opticallyactive chloroacid XV, the salt is neutralized to (-)-threo-epoxyaconiticacid (XIII), which is in turn is resolved into (+)-threo-epoxyaconiticacid (XIV) via its bis (+)-p-nitro-α-methylbenzylamine salt XXXVIII##STR37## and cleaved into the chloroacid XV by methods well-known inthe art or described hereinbefore.

The neutralization is conveniently performed by treating the mono-alkalimetal salt XXX, preferably the mono-potassium salt, with a strong acidin an appropriate solvent. Among strong acids there may be mentionedmineral acid such as hydrochloric acid, hydrobromine acid, nitric acid,perchloric acid, sulfuric acid and so forth, and organic acids such asmethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid,trifluoroacetic acid, trichloroacetic acid and so forth. Amongappropriate solvents there may be mentioned water, lower alkanol such asmethanol, ethanol, 2-propanol and the like, mixtures of water and loweralkanols, and ketones such as acetone, methylethyl ketone, diethylketoneand the like. Mineral acids and ketones are preferred. Sulfuric acid andacetone are particularly preferred.

The chlorocitric acids of the present invention of formula I and thestereoisomers, optical antipodes and pharmaceutically acceptable saltsthereof exhibit potent anorectic (appetite suppressant) activity inmammals and are thus useful in the treatment of obesity in such species.Of particular interest is (-)-threo-chlorocitric acid of formula XV andpharmaceutically acceptable salts thereof, which are significantly moreactive in the acute meal-fed assay utilizing three diets and inpyramiding dose studies as measured by reduction in food consumptionthan hydroxycitric acid, citric acid being inactive in thesedeterminations.

Similarly, in both short- and long-term chronic studies,(-)-threo-chlorocitric acid suppressed appetite and reduced body weightgain in mammals more efficaciously than hydroxycitric acid or citricacid. Even though (-)-threo-chlorocitric acid, unlike hydroxycitricacid, is devoid of metabolic activity as an inhibitor of lipid andcholesterol syntheses, carcass analysis indicated that weight loss wasdue to reduction in body lipids.

While the mechanism of action of the chlorocitric acids of formula I ofthe present invention has not been conclusively established, it appearsthat the significant mode of action in producing anorexia is a delay ingastric emptying. At comparable doses, hydroxycitric acid also delayedgastric emptying. Contributory modes of action of hydroxycitric acidinclude, however, inhibition of fatty acid and cholesterol syntheses.

The chlorocitric acids of the present invention, useful in the treatmentof obesity can be made up in the form of conventional pharmaceuticalpreparations containing, in addition to the active ingredients, carriermaterial. Such carrier material includes conventional organic orinorganic inert pharmaceutical adjuvants, additives and excipientssuitable for parenteral or enteral administration such as, for example,water, gelatin, lactose, starch, magnesium stearate, talc, vegetableoil, gums or the like. They can be administered in conventionalpharmaceutical forms, e.g., solid forms, for example, tablets, dragees,capsules, suppositories or the like; or in liquid forms, for example,suspensions or emulsions. Moreover, the pharmaceutical compositionscontaining compounds of this invention can be subjected to conventionalpharmaceutical expedients such as sterilization, and can containconventional pharmaceutical excipients such as preservatives,stabilizing agents, emulsifying agents, salts for the adjustment ofosmotic pressure or buffers. The composition can also contain othertherapeutically active materials.

A suitable pharmaceutical dosage unit can contain from about 10 to about1000 mg of (-)-threo-chlorocitric acid or its isomers. Suitableparenteral and oral dosage regimens in mammals comprise from 1 mg/kg toabout 150 mg/kg per day. However, for any particular subject, thespecific dosage regimen should be adjusted according to individual needand the professional judgement of the person administering orsupervising the administration of the aforesaid compounds. It is to beunderstood that the dosages set forth herein are exemplary only and thatthey do not to any extent, limit the scope or practice of thisinvention.

The chlorocitric acids of the present invention can also be compoundedor blended with a feed additive, premix, feed concentrate or feedadditive supplement to form a dietary admixture for administration to ananimal. A feed additive, concentrate or premix is a composition to bediluted to produce a complete feed, i.e., a composition to beadministered as a sole ration. A feed additive supplement is acomposition to be consumed directly by the animal or which can befurther diluted to produce a complete feed or which can be ingested andused as a supplement to other rations. Dietary admixtures usuallycontain a relatively large percentage of chlorocitric acids, i.e., theactive ingredient, and are conveniently prepared by adding the activeingredient to a suitable carrier and mixing, so as to assume asubstantially uniform dispersion of the anorectic in the carrier.Suitable carriers are solids that are inert with respect to the activeingredient and which may safely be ingested by the animals to betreated. Typical of such carriers are commercial animal feeds, groundcereal grains, grain by-products, plant protein concentrates (soy,peanuts, etc.), fermentation by-products, salt, limestone, inorganiccompounds, and the like or mixtures thereof. Liquid dispersions can beprepared by using water or vegetable oil, preferably including a surfaceactive agent, emulsifying agent, and the like in the liquid dispersionsuch as ethylenediamine tetraacetic acid, etc. and solubilizers. Anysuitable carrier or extender material can function as the inertingredient in the solid form of the antiobesity agent provided that itis inert to the active material and is non-toxic insofar as the animalto which it is to be administered is concerned.

The active ingredient may be blended into a mash, pellet or any desiredconfiguration with the inert carrier or extender solid material by anyconvenient technique. For example, compositions can be formed by finelygrinding or pulverizing the active ingredient and the inert ingredientusing any commercially available grinder or pulverizer with or withoutthe feed material being present. If the feed material is not presentwhen the grinding or pulverizing is effected, the resultant material canbe distributed, in accordance with the present invention, in anyconveniently available feed material. Typical animal feeds which can bemedicated with the active ingredient of this invention can containseveral ingredients, for example, they can contain high energy grainproducts such as corn, wheat, wheat red dog flour, milo, oatmeal or thelike; medium and low energy grain products such as oats, barley, wheatflour, middlings, standard middlings or the like; stabilized fats;vegetable protein such as soybean meal, corn gluten meal, peanut meal orthe like; animal protein such as fish meal, fish solubles, meat scrapsor the like; UGF (unidentified growth factor) sources and otherB-vitamin carriers such as dried milk products, dried brewers yeast,distillers dried solubles, fermentation solubles, or the like;dehydrated alfalfa meal; and various special additives such asadditional riboflavin, vitamin B₁₂, calcium pantothenate, niacin,choline, vitamin K and vitamin E or the like, as well as stabilizedvitamin A, vitamin D₃ (D-activated animal sterols); calcium andphosphorus supplements such as dicalcium phosphate, steamed bone meal,defluorinated phosphate, limestone or the like; iodized salt, manganesesulfate, zinc carbonate, an antibiotic feed supplement, methionine orits hydroxy analog and an antioxidant.

As is evident from the above, the antiobesity compositions are intendedfor oral ingestion. They can be added to the normal feed supply of thetreated animal or can be administered by other procedures, such asincorporating the same in a tablet, pill, or bolus and supplying itforcibly to the animal. The administration of the active ingredient mustbe considered in terms of the specific animal under the husbandrypractices encountered.

The actual concentration of the active ingredient in animal feed can, ofcourse, be adjusted to the individual needs and may vary over a widerange. The limiting criteria of the concentration are that the minimumconcentration is such that a sufficient amount of active ingredient isprovided to effect the desired reduction of obesity and the maximumconcentration is such that the amount of composition ingested does notresult in any untoward or undesirable side effects.

Thus, for example, a feed premix or complete feed contains sufficientactive ingredient to provide from about 0.0025% to about 1.00% by weightof the daily feed consumption. Preferably, about 0.0625% to 0.40% byweight is used. Most preferably about 0.125% by weight is used.

The present invention may be more clearly illustrated by the followingexamples. All temperatures are stated in degrees Centigrade.

EXAMPLE 1 (±)-Threo-Chlorocitric acid

a. Hypochlorination Method.

174 g. (1.0 mole) of trans-aconitic acid was added portionwise to astirred solution of 120 g. (3.0 mol) of sodium hydroxide in 400 ml. ofwater. Throughout the addition, the reaction temperature was maintainedat ca. 25° by adding ice to the mixture as needed (total ca. 350 g.).When the acid had completely dissolved, the solution was at pH 7.5. (Inother experiments, the solution was adjusted to pH 7.5 by the additionof either 2 N sodium hydroxide or trans-aconitic acid as needed.) Thesolution was then transferred to a 3-liter, 3-necked round bottom flaskfitted with a mechanical stirrer, a gas inlet tube (fritted disc type),a thermometer and a gas outlet. The gas outlet was connected via rubbertubing to a Pasteur pipette which was partially submerged in a column ofwater in a graduated cylinder. This was used to monitor the rate ofgases escaping the reaction vessel. The reaction mixture was cooled to5° and purged with argon. Chlorine gas was then added to the rapidlystirred mixture as fast as it could be consumed without excess gasexiting the gas outlet. Initially the temperature rose rapidly to ca.15° but it was maintained at 10°-15° throughout the reaction by externalcooling (ice-acetone bath). After 20-30 minutes, the absorption rate ofchlorine gas diminished rapidly and the solution became typically green.When no more gas was absorbed (as indicated by the gas exit flow) theaddition of chlorine gas was stopped and the mixture was stirred at 10°for another 10 minutes. Excess chlorine gas was then purged by bubblingargon gas through the reaction mixture.

b. Epoxide Cleavage Method.

To a solution of 123 g (0.5 mol) of mono-potassium(±)-threo-epoxyaconitic acid, monohydrate, 28 g (0.375 mol) of potassiumchloride and 120 ml of water was added 88 ml (ca. 1.06 mol) of conchydrochloric acid. The reaction mixture was heated at 70° for 15 hours,allowed to cool to room temperature and concentrated at 50° and 0.5 to1.0 mm of mercury. Ethyl acetate (250 ml) was added and the mixture wasagitated at 40°. The precipitated potassium chloride was collected andwashed with 350 ml portion of ethyl acetate. The filtrate was evaporatedto dryness at 50° under water aspirator pressure. The residue wasdissolved in ethyl acetate, treated with anhydrous magnesium sulfate andfiltered through a pad of Celite Filter-Aid. The filter cake was washedwith ethyl acetate. Carbon tetrachloride was added to the filtrate, withstirring, until the cloud point was reached. The turbid mixture wasseeded with authentic crystalline (±)-threo-chlorocitric acid,monohydrate, and the mixture was stirred for 2 hours at room temperatureand allowed to stand for 16 hours in a refrigerator. The precipitate wascollected, washed with carbon tetrachloride, ethyl acetate (3:1) and airdried to afford 70.9 g of product, m.p. 74°-76°, having a neutralizationequivalent of 81.58 of g/equiv (theoretical neutralization equiv 81.52).

The mother liquors were evaporated to dryness at 50° and water aspiratorpressure. The residue was dissolved in 125 ml of ethyl acetate andtreated with carbon tetrachloride as described above to give 20.7 g ofproduct, m.p. 74°-76°, having a neutralization equivalent of 82.00.

A 91.0 g-portion of the combined first and second crops of the productwas dissolved in 250 ml of ethyl acetate and treated with ca. 500 ml ofcarbon tetrachloride. The turbid solution was seeded with crystalline(±)-threo-chlorocitric acid monohydrate and stored in a refrigeratorovernight. The precipitate was collected, washed with carbontetrachloride and ethyl acetate as described above and air dried toyield 84.3 g of purified product, m.p. 74°-76°, having a neutralizationequivalent of 81.5.

Calcd for C₆ H₇ ClO₇.H₂ O: C, 29.46; H, 3.71; Cl, 14.50; H₂ O, 7.36.Found: C, 29.27; H, 3.72; Cl, 14.23; H₂ O, 7.43.

The reaction was then acidified using 175 ml conc. hydrochloric acid(2.1 mol) and then it was heated at 70° for 1 hour to hydrolyze theβ-lactone. The solution was then concentrated in vacuo to dryness andthe residue was triturated with warm ethyl acetate (3×500 ml). Thecombined extracts were filtered to remove residual sodium chloride andthen were dried over magnesium sulfate. Evaporation of the solvent gavea solid which was redissolved in ethyl acetate (500 ml) and the solutionwas diluted to the cloud point with carbon tetrachloride. After themixture was stirred overnight at room temperature, the solid which hadformed was recovered by filtration to give 102.0 g of(±)-threo-chlorocitric acid (dried in vacuo to constant weight), mp96°-101°; neutralization equivalent 75.81 g/eg (Theory 75.52 g/eg).

The mother liquors were concentrated to dryness and were thencrystallized as above to give an addition 60.7 g of pure(±)-threo-chlorocitric acid.

The analytically pure material was obtained by recrystallization fromethyl acetate-carbon tetrachloride and had mp 76°-77° as themonohydrate.

EXAMPLE 2 (±)-erythro-Chlorocitric acid.

74.0 g of cis-aconitic anhydride (0.474 mol) was dissolved in 200 ml ofwater containing 100 g ice. A solution of sodium hydroxide (46.25 g;1.15 mol) in 100 ml of water was added slowly with stirring. Thereaction temperature was held below 20° by the simultaneous addition of200 g ice. When the base addition was completed, 15.25 g sodiumtungstate dihydrate, followed by 55.5 ml of 30% hydrogen peroxide wasadded to the mixture. The oxidation was initiated by gently warming thestirred solution to 23°. The external heat source was then removedwhereupon the heat of reaction caused the mixture temperature to slowlyclimb to 51.5° over 25 minutes after which it started to decline. Afterstirring an additional 7-10 minutes, the reaction mixture was treatedwith 150 ml conc hydrochloric acid and 150 g sodium chloride and heatedat 75° for 15 minutes. In this way the intermediate(±)-erythro-epoxyaconitic acid was converted in situ to(±)-erythro-chlorocitric acid. After the reaction mixture was cooled toroom temperature, 2.3 g sodium bisulfite₃ was added to destroy residualhydrogen peroxide. The solution was then transferred to a liquid-liquidextraction apparatus and was continuously extracted using ether.

The first extract collected after 21 hours was dried over magnesiumsulfate and concentrated in vacuo to give 73.0 g of solid crudechlorocitric acid contaminated with ca 8% trans-aconitic acid.Crystallization of this material twice from ethyl acetate-carbontetrachloride finished essentially pure (±)-erythro-chlorocitric acid,mp 162°-164°.

A second extract collected after an additional 48 hrs was worked up asabove to give an additional 18.5 g of the chlorocitric acid, mp163°-165°.

EXAMPLE 3 (±)-threo-Chlorocitric acid, β-lactone

A solution of trisodium trans-aconitate prepared from 58.0 gtrans-aconitic acid (0.33 mol) and sodium hydroxide (40 g; 1.0 mol) in300 ml of water was cooled to 5° and chlorinated as in Example 1. Theresulting solution of disodium chlorocitric acid, β-lactone was purgedfree of excess chlorine gas and then was treated with 60 ml 12 Nhydrochloricacid (0.72 equiv). The reaction mixture was extracted (3times) with ethyl acetate and the extracts were combined and dried overmagnesium sulfate. The ethyl acetate solution was then concentrated invacuo to ca 200 ml and was diluted to the cloud point with carbontetrachloride. The resulting crystalline material was collected byfiltration to give a total of 41.5 g of pure (±)-threo-chlorocitricacid, β-lactone, mp 162°-164°.

EXAMPLE 4 Resolution of (±)-erythro-chlorocitric acid

30 g of (±)-erythro-chlorocitric acid (132.4 mmol) was dissolved in 175ml of a methanol-water mixture (49:1). The solution was cooled to 15°and 39.5 g (-)-p-nitro-α-methyl-benzylamine (238 mmol) in 75 ml of thesame methanol-water mixture was added over 2-3 minutes. The mixture wasstirred at room temperature for 18 hours during which time a white solidformed. The solids were collected by filtration and then were washedwith ethanol and with ether to give 24.0 g of partially resolved(+)-erythro-chlorocitric acid, bis(-)-p-nitro-α-methylbenzylamine salt.Attempts to further purify this salt by normal recrystallizationprocedures were frustrated by the heat required to dissolve the salt.This caused extensive conversion to the correspondingerythro-hydroxycitric acid.

The impure salt was then split in the following manner: hydrogenchloride gas was bubbled through a stirred suspension of finely dividedsalt (27.1 g) in ether for 30 minutes. The resulting solid(-)-p-nitro-α-methylbenzylamine hydrochloride (19.9 g, mp 247°-249°) wasremoved by filtration and the filtrate was concentrated in vacuo to give10.9 g of partially resolved (+)-erythro-chlorocitric acid as an oil (ca65% optical purity). The crude chlorocitric acid was treated withdiazomethane and the resulting trimethyl chlorocitrate was analyzed bynuclear magnetic resonance spectroscopy using chiral shift reagents todetermine optical purity.

The recovered (-)-p-nitro-α-methylbenzylamine hydrochloride salt waspartitioned between dichloromethane and 1 N sodium hydroxide. The aminerecovered from this process (15.6 g) in 40 ml methanol-water (49:1) wasadded to a solution of the crude (+)-erythro-chlorocitric acid in 40 mlmethanol-water (49:1) and the mixture, after stirring several hours,deposited 18.1 g of enriched bis-amine chlorocitrate salt.

The salt was again split using ethereal hydrogen chloride and was thenreformed in the manner described above. This gave 14.4 g of thebis-amine chlorocitric acid. The (+)-erythro-chlorocitric acid recoveredfrom the latest salt was recrystallized from ethyl acetate-carbontetrachloride to give 4.3 g of material (29% chemical yield) which was85% optically pure.

The mother liquors of the solution of the partially resolved(+)-erythrochlorocitric acid, bis-(-)-p-nitro-α-methylbenzylamine saltwere treated with 13 ml (0.52 mol) of conc hydrochloric acid andevaporated to dryness. 1,2-Dimethoxyethane was added and the solutionwas evaporated to dryness. Ether was added to the residue, the mixturewas stirred and the precipitate was collected to afford 29.2 g of(-)-p-nitro-α-methylbenzylamine hydrochloride.

The filtrate was concentrated to dryness and the residue, rich in(-)-erythrochlorocitric acid, was dissolved in 90 ml of methanol-water(49:1). A solution of 25.3 g (0.125 mol) of(+)-p-nitro-α-methylbenzylamine and 50 ml of methanol-water (49:1) wasadded. The resulting mixture was stirred at room temperature for 20hours and the precipitate was collected to afford 15.0 g ofbis-(+)-p-nitro-α-methylbanzylamine salt enriched in(-)-erythro-chlorocitric acid. The salt was suspended in 185 ml of etherand hydrogen chloride was added to the suspension over a period of 30min. The precipitated (+)-p-nitro-α-methylbenzylamine hydrochloride(10.7 g) was collected on a filter. The filtrate was concentrated todryness and the residue was dissolved in 40 ml of methanol-water (49:1).To the methanol-water solution was added a solution of 8.9 g (0.54 mol)of (+)-p-nitro-α-methylbenzylamine and 15 ml of methanol-water (49:1)and the solution was stirred at room temperature for ca. 2.5 hours. Theprecipitate, (11.75 ) enriched in (-)-erythro-chlorocitric acidbis-(+)-p-nitro-α-methylbenzylamine salt, was suspended in ether and theresulting suspension was saturated with hydrogen chloride. Theprecipitated (+)-p-nitro-α-methylbenzylamine hydrochloride (8.09 g) wascollected and the filtrate was concentrated. Recrystallization of theresidue from ethyl acetate-carbon tetrachloride gave 3.9 g of(-)-erythro-chlorocitric acid (85% optical purity).

EXAMPLE 5 (-)-threo-Chlorocitric acid

105 g (0.5 mol) (+)-threo-epoxyaconitic acid monohydrate was dissolvedin 150 ml of water containing 43 ml (0.516 mol) conc hydrochloric acid.Sodium chloride (50 g) was added to the stirred solution and the mixturewas heated at 70° for 12 hours. The solution was then evaporated todryness in vacuo (ca 1 mm; 50°) and the residue was triturated with 400ml warm ethyl acetate. The mixture was filtered free of sodium chlorideand the filtrate was decolorized (acid washed charcoal), dried overmagnesium sulfate and concentrated in vacuo to a crystalline mass. Theresidue was dissolved in 250 ml ethyl acetate and the solution wasbrought to a cloud point using carbon tetrachloride. The mixture wasstirred several hours at room temperature and then was chilled in arefrigerator overnight. The solids were removed by filtration to give68.5 g of (-)-threo-chlorocitric acid, mp 138°-140° [α]_(D) ²⁵ -6.60°(c, 2.0, H₂ O). An additional 20.2 g of material [mp 138°-140°; [α]_(D)²⁵ -6.6°] were recovered from the mother liquors.

The analytically pure specimen was obtained by recrystallization fromethyl acetate-carbon tetrachloride, mp 140.5°-142.0°; [α]₂₅ ^(D) -7.05°(c, 2.0, H₂ O). EXAMPLE 6

(+)-threo-Chlorocitric acid

(-)-threo-Epoxyaconitic acid monohydrate (105 g, 0.5 mol) was dissolvedin 150 ml of water containing 43.0 ml conc. hydrochloric acid (0.516mol). To the stirred solution 50 g of sodium chloride was added and themixture was heated at 70° for 12 hours. The reaction was worked up as inExample 5 to give (+)-threo-chlorocitric acid in two crops:

Crop #1: mp 138°-140°; [α]_(D) ²⁵ +6.65° (C, 2.0, H₂ O); 55.2 g.

Crop #2: mp 138°-140°; [α]_(D) ²⁵ +6.55° (C, 2.0, H₂ O); 23.0 g.

Recrystallization of the solid from ethyl acetate-carbon tetrachloridegave the analytically pure material, mp 140.5-142°; [α]_(D) ²⁵ +6.9° (C,2.0, H₂ O).

EXAMPLE 7 (-)-erythro-Epoxyaconitic acid

A solution of 49.2 g(-)-threo-1-mesyloxy-2-hydroxy-1,2,3-propanetricarboxylic acid,trimethyl ester (0.15 mole) in 340 ml acetic acid containing 170 ml conchydrochloric acid was heated at 80° for 23 hours. The solvents wereremoved in vacuo and the crude product (mostly1-mesyloxy-2-hydroxy-2-carbomethoxy-1,3-propanedicarboxylic acid) wasevaporated several times from glyme to eliminate remaining acetic acidand hydrochloric acid. The residue was dissolved in 50 ml of water andthe solution was added at a rapid dropwise rate with stirring to 157.5ml of sodium hydroxide (0.63 mol) initially cooled to 5° (thetemperature rose to ca 27° during the addition). The reaction wasstirred at room temperature for 30 minutes to ensure complete hydrolysisof the remaining ester function as well as formation of the oxiranering. The reaction mixture was cooled to 10° and then was adjusted to pH7.5 using a total of 5.5 ml 3.6 N sulfuric acid, whereupon 12.5 ml ofconc. sulfuric acid (0.45 eg) was added carefully to convert thetrisodium epoxyaconitic acid into its free acid. The solution wasconcentrated in vacuo at ca. 30° until crystals of sodium sulfatedecahydrate began to deposit and then it was diluted with 300 mlacetone. The inorganic salt was removed by filtration and the filtratewas evaporated to dryness under reduced pressure. A solution of thereaction product in tetrahydrofuran (300 ml) was filtered to removeresidual sodium sulfate, dried over magnesium sulfate, decolorized(Norit SG acid washed charcoal) and concentrated to dryness in vacuo togive a solid residue. Crystallization of the crude product from ethylacetate-carbon tetrachloride (400:300 ml) furnished 17.7 g of(-)-erythro-epoxyaconitic acid, mp 169°-172°. This sample wascontaminated by a minor amount of sodium methylsulfonate which wasremoved by placing the material in a thimble in a Sohxlet apparatus andextracting it using ether (1.5 l) over 1.5 hours. The ether extract wasconcentrated in vacuo and the solid residue was crystallized from ethylacetate-carbon tetrachloride (325:125 ml) to give 13.0 g of pure(-)-erythro-epoxyaconitic acid mp 179.5°-181°; [α]_(D) ²⁵ -52.4° (C,1.0, H₂ O). Concentration of the mother liquor afforded a second crop,2.1 g; mp 179°-181°.

EXAMPLE 8 (+)-erythro-Epoxyaconitic acid

Using the procedure described in Example 7,112.7 g(+)-threo-1-mesyloxy-2-hydroxy-1,2,3-propanetricarboxylic acid,trimethyl ester was converted into 36.2 g (+)-erythro-epoxyaconiticacid, mp 173°-175°. This material was again contaminated with a trace ofsodium methanesulfonate which was removed as before to give theanalytically pure epoxytriacid, mp 179.5°-181°; [α]_(D) ²⁵ +52.6° (C,1.0, H₂ O).

EXAMPLE 9 (-)-erythro-Chlorocitric acid

A solution of 8.1 g (+)-erythro-epoxyaconitic acid (42.6 mmol) in 43 ml1 N hydrochloric acid (43 mmol) containing 15 g sodium chloride washeated at 78° for 25 minutes. At the end of this time, examination ofthe reaction mixture by nuclear magnetic resonance spectroscopy showed asignificant amount of starting epoxide remaining and so the reaction washeated an additional 20 minutes at 80° to complete the reaction.Evaporation of the solvent in vacuo left a residue consisting of crude(-)-erythro-chlorocitric acid and sodium chloride. The organic materialwas dissolved in glyme and the resulting solution was filtered to removesodium chloride and then was dried over magnesium sulfate andconcentrated under reduced pressure. Crystallization of the crudereaction product from ethyl acetate-carbon tetrachloride afforded 7.4 gof essentially pure (-)-erythro-chlorocitric acid. Recrystallizationfurnished 5.4 g (dried to constant weight) of pure triacid, mp133.5°-135°; [α]_(D) ²⁵ -2.2° (C, 2.0, H₂ O).

EXAMPLE 10 (+)-erythro-Chlorocitric acid

(-)-erythro-Epoxyaconitic acid (9.5 g) was converted into 7.6 g of(+)-erythrochlorocitric acid (mp 132°-134°) by a procedure essentiallyidentical to that described in Example 9. Recrystallization of thechlorocitric acid thus obtained afforded 5.3 g of analytically purematerial, mp 133.5°-135°; [α]_(D) ²⁵ +2.2° (C, 2.0, H₂ O).

EXAMPLE 11 (±)-threo-Epoxyaconitic acid

trans-Aconitic acid (87.0 g, 0.5 mol) was added portionwise to asolution of 60.0 g of sodium hydroxide (1.5 mol) in 200 ml of water. 150g of ice was added as needed to moderate the heat of neutralization. Themixture was cooled to 0°-5° and was chlorinated as in Example 1. Theresulting solution of (±)-threo-chlorocitric acid, lactone disodium salt(purged free of excess chlorine gas) was cooled to -10° and then wastreated with 40.0 g (1.0 mol) sodium hydroxide. The solution was stirredrapidly as the pellets dissolved and the reaction temperature wasmoderated by external cooling so that it did not exceed 20°. The mixturewas stirred at 20° for 20 minutes and then 42 ml of conc sulfuric acid(1.512 equiv) was added dropwise with cooling. The solution wastransferred to a liquid-liquid extractor and was extracted over 2-3 daysusing diethyl ether. The ether extract was dried over magnesium sulfateand concentrated in vacuo. The resulting solid was crystallized fromethyl acetate-carbon tetrachloride to give 67.8 g of (±)-threo-epoxyacid, mp 169°-172°. A second crop (8.85; mp 167°-170°) was collectedfrom the mother liquors.

EXAMPLE 12 (-)-threo-Hydroxycitric acid

(-)-threo-Hydroxycitric acid ethylenediamine salt (26.8 g, 0.1mole) wasadded in one portion to a stirred mixture of trifluoroacetic acid (130ml) and water (5.0 ml) previously cooled to 5°-10°. Within severalminutes the salt had essentially all dissolved and crystals of(-)-threo-hydroxycitric acid had begun to form in the mixture. After 30minutes the solids were collected by filtration and were washed withtrifluoroacetic acid (3×50 ml). The motor liquors were discarded and thesolid was washed with ether and dried to give 20.1 g of the triacid, mp154.5°-155.5°. This material was recrystallized by dissolving 20.1 g oftriacid in 18 ml of water at 15° and diluting the solution in oneportion with 100 ml trifluoroacetic acid. The mixture was chilled to-10° in an acetone ice bath and the resulting crystalline material wasrecovered to give 19.2 g of (-)-threo-hydroxycitric acid, mp156.5°-158°; [α]_(D).sup. 25 -9.9° (C, 1.0, H₂ O).

EXAMPLE 13 Comparative anorectic activity of (±)-threo-chlorocitric acidand trisodium (-)-threo-hydroxycitric acid

Charles River female rats (Charles River Breeding Laboratories,Wilmington, Mass.), weighing 150 to 175 g, were individually housed inwire-bottomed cages in a temperature-regulated (22° C.) light-controlledroom (12 hr light, 6 a.m. to 6 p.m. and 12 hr dark, 6 p.m. to 6 a.m.).Animals were fasted 48 hr, then meal-fed the 70% glucose diet describedby A. C. Sullivan, et al., J. Nutrition, 101, 265 (1971) from 8 to 11a.m. Following 5 to 13 days alimentation on the meal-feeding regimen,rats were dosed with the appropriate compounds orally by intubation 1/2hr before the 3 hr meal. Food cups were weighed immediately after themeal. The control group consists of 31 rats while each drug treatedgroup consists of 5 to 12 rats.

The G-70 diet consisted of 70% glucose, 23% vitamin-free casein, 5%Phillips and Hart salt mixture IV, 1% corn oil, 1% complete vitaminmixture, and 40 g/kg cellulose. To ensure complete uniformity, all dietswere mixed with a twen-shell dry blender equipped with an itensifier bar(Patterson-Kelley Co., East Stroudsburg, Pa.).

    ______________________________________                                        RESULTS                                                                               Food Consumption                                                                (±)-threo-Chlorocitric                                                                    Trisodium (-)-                                       Concentration.sup.a                                                                     Acid           threo-hydroxycitrate                                 mmoles/kg           % of              % of                                    body weight                                                                             g         control  g        control                                 ______________________________________                                        Control   11.2 ± 0.5.sup.b                                                                     100      11.2 ± 0.5                                                                          100                                     2.63      3.8 ± 0.3**                                                                          34       8.9 ± 0.8**                                                                         79                                      1.32      3.1 ± 0.4**                                                                          28       9.9 ± 1.0                                                                           88                                      0.66      3.9 ± 0.3**                                                                          35       9.0 ± 0.6**                                                                         80                                      0.33      5.7 ± 0.5**                                                                          51       11.5 ± 0.7                                                                          103                                     0.17      6.8 ± 0.7**                                                                          61                                                        0.08      6.4 ± 0.6**                                                                          57                                                        ______________________________________                                         .sup.a Compounds were dissolved in water and administered orally at the       indicated concentrations.                                                     .sup.b Each value is the mean ± S.E.                                       **p ≦ 0.01                                                        

EXAMPLE 14 Effect to stereoisomers of chlorocitric acid on foodconsumption

Charles River female rats (Charles River Breeding Laboratories,Wilmington, Mass.), weighing 130 to 150 g, were individually housed inwire-bottomed cages in a temperature-regulated (22° C.) light-controlledroom (12 hr light, 6 a.m. to 6 p.m. and 12 hr dark, 6 p.m. to 6 a.m.).Animals were fasted 48 hr, then meal-fed the 70% glucose diet describedby A. C. Sullivan, et al., J. Nutrition, 101, 265 (1971) from 8 to 11a.m. Following 5 to 12 days alimentation on the meal-feeding regimen,rats were dosed with the appropriate compounds orally by intubation 1/2hr before the 3 hr meal. Food cups were weighed immediately after themeal. The control group consisted of 5 to 10 rats, while theexperimental group consisted of 4 to 6 rats.

    ______________________________________                                        RESULTS                                                                                   Food Consumption                                                               2.63 mmoles/kg                                                                           0.66 mmoles/kg                                                    body weight body weight                                                                    % of           % of                                                           con-           con-                                  Treatment     g          trol   g       trol                                  ______________________________________                                        Control       13.5 ± 0.8.sup.a                                                                      100    8.2 ± 1.2                                                                          100                                   Citric Acid   12.7 ± 1.7                                                                            94     --      --                                    (+)-threo-Chlorocitric                                                        acid           5.1 ± 1.2***                                                                         38     6.8 ± 1.0                                                                          83                                    (-)-threo-Chlorocitric                                                        acid           2.2 ± 0.5***                                                                         16      4.8 ± 0.7*                                                                        59                                    (-)-erythro-Chlorocitric                                                      acid           7.2 ± 1.5***                                                                         53     7.0 ± 1.9                                                                          85                                    (+)-erythro-Chlorocitric                                                      acid          10.5 ± 0.8*                                                                           78     8.1 ± 1.6                                                                          99                                    ______________________________________                                         .sup.a Each value is the mean ± S.E.                                       *P ≦ 0.05                                                              ***P ≦ 0.001                                                      

EXAMPLE 15 The effect of (+)-threo-chlorocitric acid and(-)-threo-chlorocitric acid on food consumption of three diets

Charles River female rats (Charles River Breeding Laboratories,Wilmington, Mass.), weighing 170 to 200 g, were individually housed inwire-bottomed cages in a temperature-regulated (22° C.) light-controlledroom (12 hr light, 6 a.m. to 6 p.m. and 12 hr dark, 6 p.m. to 6 a.m.).Animals were fasted 48 hr, then meal-fed the indicated diets from 8 to11 a.m. Following 5 to 12 days alimentation on the meal-feeding regimen,rats were dosed with the appropriate compounds orally by intubation 1/2hr before the 3 hr meal. Food cups were weighed immediately after the1st, 2nd and 3rd hr of the meal. The control groups consisted of 8 to 10rats and the experimental group considered of 5 to 6 rats.

    __________________________________________________________________________    RESULTS                                                                                             Dose     Food Intake (g).sup.a                          Treatment     Diet    mmoles (mg)/kg                                                                         1st hr                                                                              2nd hr                                                                              3rd hr                                                                              Total                        __________________________________________________________________________    Control       70% Glucose                                                                           --       5.8 ± 0.7.sup.b                                                                  4.8 ± 0.5                                                                        1.9 ± 0.5                                                                        12.6 ± 1.2                (+)-threo-Chlorocitric acid                                                                 70% Glucose                                                                           0.66 (150)                                                                             3.2 ± 0.5*                                                                       5.1 ± 1.3                                                                        1.7 ± 0.3                                                                        10.1 ± 1.7                              70% Glucose                                                                           0.33 (75)                                                                              6.2 ±1.8                                                                         2.4 ±0.8*                                                                        2.4 ±0.5                                                                         11.0 ±2.3                 (-)-threo-Chlorocitric acid                                                                 70% Glucose                                                                           0.66 (150)                                                                             2.7 ± 0.4*                                                                       1.7 ± 0.3*                                                                       2.3 ± 0.5                                                                         6.7 ± 0.7**                            70% Glucose                                                                           0.33 (75)                                                                              2.2 ± 0.5**                                                                      2.2 ± 0.5**                                                                      3.8 ± 0.4*                                                                        8.2 ± 1.1*               Control       Chow    --       7.7 ± 0.7                                                                        3.0 ± 0.5                                                                        3.5 ± 0.5                                                                        14.1 ± 0.9                (+)-threo-Chlorocitric acid                                                                 Chow    0.66 (150)                                                                             7.7 ± 0.5                                                                        3.9 ± 0.5                                                                        1.8 ± 0.9                                                                        13.4 ± 0.9                              Chow    0.33 (75)                                                                              7.4 ± 0.6                                                                        2.6 ±  0.7                                                                       2.2 ± 0.5                                                                        12.2 ± 0.4                (-)-threo-Chlorocitric acid                                                                 Chow    0.66 (150)                                                                             3.6 ± 0.9**                                                                      1.4 ± 0.2*                                                                       1.9 ± 0.4*                                                                        6.9 ± 1.2**                            Chow    0.33 (75)                                                                              4.2 ± 1.4*                                                                       1.6 ± 0.4                                                                        2.3 ± 0.7                                                                         8.1 ± 2.1*               Control       20% Corn Oil                                                                          --       6.8 ± 1.0                                                                        3.1 ± 0.7                                                                        2.9 ± 0.8                                                                        12.7 ± 0.7                (+)-threo-Chlorocitric acid                                                                 20% Corn Oil                                                                          0.66 (150)                                                                             6.7 ± 1.6                                                                        3.1 ± 0.6                                                                        2.2 ± 0.7                                                                        12.3 ± 1.6                              20% Corn Oil                                                                          0.33 (75)                                                                              7.1 ± 1.2                                                                        3.9 ± 0.5                                                                        3.2 ± 0.6                                                                        14.2 ± 1.2                (-)-threo-Chlorocitric acid                                                                 20% Corn Oil                                                                          0.66 (150)                                                                             3.6 ± 0.6*                                                                       2.2 ± 0.4                                                                        1.3 ± 0.4                                                                         7.1 ± 0.9*                             20% Corn Oil                                                                          0.33 (75)                                                                              4.0 ± 0.6                                                                        1.6 ± 1.1                                                                        1.3 ± 0.5                                                                         6.8 ± 1.6**              __________________________________________________________________________     .sup.a Food consumption was measured by weighing food cups at the             indicated times, following the initiation of the meal.                        .sup.b Each value is the mean ± S.E.                                       *P ≦ 0.05                                                              **P ≦ 0.01                                                        

EXAMPLE 16 Anorectic activities or stereoisomers or chlorocitric acid

Charles River female rats (Charles River Breeding Laboratories,Wilmington, Mass.), weighing 196 to 210 g, were individually housed inwire-bottomed cages in a temperature-regulated (22° C.) light-controlledroom (12 hr light, 6 a.m. to 6 p.m. and 12 hr dark, 6 p.m. to 6 a.m.).Animals were fasted 48 hr, then meal-fed a 70% glucose diet from 8 to 11a.m. Following 5 to 12 days alimentation on the meal-feeding regimen,rats were dosed with the appropriate compounds orally by intubation 1/2hr before the 3 hr meal for 3 days. The total food consumption and bodyweight gained during the 3 day period were determined. The control groupconsisted of 16 rats, while the experimental group consists of 8 rats.

    __________________________________________________________________________    RESULTS                                                                                            3-Day      3-Day                                                              Food Consumption                                                                         Body Weight Gain                                             Dose         % of        % of initial                          Treatment      mmoles/kg                                                                           g      control                                                                           g       body weight                           __________________________________________________________________________    Control        --    28.6 ± 1.1.sup.a                                                                  100 -0.2 ± 1.1                                                                         -0.1                                  (+)-threo-Chlorocitric acid                                                                  1.32  26.5 ± 1.3                                                                        93  -3.5 ± 1.2                                                                         -1.7                                  (-)-threo-Chlorocitric acid                                                                  1.32  10.5 ± 1.3**                                                                      37  -22.4 ± 2.3**                                                                      -11.0                                 (-)-erythro-Chlorocitric acid                                                                1.32  24.5 ± 1.8*                                                                       86  -4.8 ± 3.2                                                                         -2.4                                  (+)-erythro-Chlorocitric acid                                                                1.32  31.8 ± 2.3                                                                        111 -1.5 ± 2.3                                                                         -0.7                                  __________________________________________________________________________     .sup.a Each value is the mean ± S.E.                                       *P ≦ 0.05                                                              **P ≦ 0.01                                                        

EXAMPLE 17 Anoretic effect of (±)-threo-chlorocitric acid, (-)-threochlorocitric acid and (+)-threo-chlorocitric acid in 8 hour meal-fedzucker rats

Lean (Fa/-) Zucker rats and obese (fa/fa) Zucker rats of both sexes wereindividually housed in wire-bottomed cages in a temperature-regulated(22° C.) light-controlled room (12 hr light, 6 a.m. to 6 p.m. and 12 hrdark, 6 p.m. to 6 a.m.). The animals were fed a high carbohydrate (70%glucose) diet ad libitum for 13 days, dosed with the indicated compoundsby oral intubation for 9 days and allowed to recover for 3 days. Theanimals were then meal fed from 8 a.m. to 4 p.m. for a period of 5 days,following which they were continued on the same meal schedule for anadditional 6 days. During the 6-day period, the animals wereadministered the indicated compounds by oral intubation at 8 a.m. and 12p.m. Food cups were weighed immediately after the meal.

    __________________________________________________________________________    RESULTS                                                                                    Concen-                                                                       tration                                                                       mmoles/kg                                                                           Daily Food Consumption (g)                                          Geno-                                                                             body  Pretreat-                                                                             Treatment Period (days)                            Treatment                                                                              type.sup.a                                                                         weight                                                                             ment 0  1     2     3     4     5     6                    __________________________________________________________________________    Control  Fa/-                                                                              --    10.9 ± 0.6.sup.b                                                                  10.6 ± 1.2                                                                        11.2 ± 1.0                                                                       12.5 ± 0.7                                                                       11.7 ± 1.0                                                                       13.4                                                                                14.2 ± 1.3        Citric acid                                                                            Fa/-                                                                              1.32  11.0 ± 0.8                                                                        10.6 ± 1.2                                                                        10.0 ± 1.2                                                                       12.7 ± 1.0                                                                       11.9 ± 0.7                                                                       12.6                                                                                13.1 ± 0.9        (±)-threo-Chloro-                                                                   Fa/-                                                                              1.32  12.3 ± 0.7                                                                        11.6 ± 0.8                                                                        7.5 ± 0.8**                                                                      8.6 ± 1.3**                                                                      6.9 ± 1.0**                                                                      6.1 ± 0.7**                                                                      7.2 ± 1.0**       citric acid                                                                   (-)-threo-Chloro-                                                                      Fa/-                                                                              0.66  10.8 ± 1.0                                                                        12.5 ± 1.1                                                                        7.0 ± 0.9**                                                                      7.2 ± 0.7**                                                                      5.4 ± 1.1**                                                                      7.3 ± 1.5**                                                                      7.5 ± 1.3**       citric acid                                                                   (+)-threo-Chloro-                                                                      Fa/-                                                                              0.66  10.6 ± 0.8                                                                        10.1 ± 0.8                                                                        8.7 ± 0.3**                                                                      11.7 ± 0.7                                                                       9.8 ± 0.9                                                                        12.8                                                                                11.4 ± 0.7        citric acid                                                                   Control  fa/fa                                                                             --    11.1 ± 0.9                                                                        10.6 ± 0.5                                                                        12.4 ± 0.9                                                                       14.3 ± 1.3                                                                       12.4 ± 1.1                                                                       13.2                                                                                14.3 ± 1.2        Citric Acid                                                                            fa/fa                                                                             1.32  13.4 ± 0.8                                                                        11.4 ± 0.8                                                                        13.8 ± 1.2                                                                       16.5 ± 1.0                                                                       14.8 ± 0.9                                                                       15.7                                                                                16.0 ± 1.0        (±)-threo-Chloro-                                                                   fa/fa                                                                             1.32  14.3 ± 0.8**                                                                      13.1 ± 0.9**                                                                      7.8 ± 0.8**                                                                      6.6 ± 0.2**                                                                      5.9 ± 0.8**                                                                      4.6 ± 1.1**                                                                      5.3 ± 1.3**       citric acid                                                                   (-)-threo-Chloro-                                                                      fa/fa                                                                             0.66  13.1 ± 1.1                                                                        10.7 ± 0.7                                                                        5.9 ± 1.5**                                                                      5.8 ± 0.9**                                                                      5.5 ± 0.9**                                                                      4.9 ±                                                                            5.1 ± 1.1**       citric acid                                                                   (+)-threo-Chloro-                                                                      fa/fa                                                                             0.66  14.3 ± 1.4                                                                        10.3 ± 0.4                                                                        8.8 ± 0.8**                                                                      11.3 ± 1.0                                                                       11.6 ± 1.2                                                                       12.3                                                                                14.2 ± 1.3        citric acid                                                                   __________________________________________________________________________     .sup.a Fa/, lean rats;                                                        fa/fa, obese rats.                                                            .sup.b Each value is the mean ± S.E.                                       *P ≦ 0.01                                                         

EXAMPLE 18 Effect of (±)-threo-chlorocitric acid, (-)-threo-chlorocitricacid and (+)-threo-chlorocitric acid on body weight gain in 8 hour mealfed zucker rats

Lean (Fa/-) Zucker rats and obese (fa/fa) Zucker rats of both sexes wereindividually housed in wire-bottomed cages in a temperature-regulated(22° C.) light-controlled room (12 hr light, 6 a.m. to 6 p.m. and 12 hrdark, 6 p.m. to 6 a.m.). The animals were fed a high carbohydrate (70%glucose) diet ad libitum for 13 days, dosed with the indicated compoundsby oral intubation for 9 days and allowed to recover for 3 days. Theanimals were then meal fed from 8 a.m. to 4 p.m. for a period of 5 days,following which they were continued on the same meal schedule for anadditional 6 days. During the 6-day period, the animals wereadministered the indicated compounds by oral intubation at 8 a.m. and 12p.m. Body weight gain during the 6-day period was determined.

    ______________________________________                                        RESULTS                                                                                          Concentration                                                                 mmoles/kg                                                             Geno-   body weight Body Weight Gain                               Treatment  type.sup.a                                                                            b.i.d.      g                                              ______________________________________                                        Control    Fa/-    --          9.5 ± 2.2.sup.b                             Citric Acid                                                                              Fa/-    1.32        2.8 ± 3.7                                   (±)-threo-Chloro-                                                                     Fa/-    1.32        -22.9 ± 2.9*                                citric acid                                                                   (-)-threo-Chloro-                                                                        Fa/-    0.66        -20.7 ± 2.2*                                citric acid                                                                   (+)-threo-Chloro-                                                                        Fa/-    0.66        2.5 ± 2.3*                                  citric acid                                                                   Control    fa/fa   --          5.8 ± 4.2                                   Citric Acid                                                                              fa/fa   1.32        3.4 ± 1.6                                   (±)-threo-Chloro-                                                                     fa/fa   1.32        -27.9 ± 4.6*                                citric acid                                                                   (-)-threo-Chloro-                                                                        fa/fa   0.66        -33.5 ±3.1*                                 citric acid                                                                   (+)-threo-Chloro-                                                                        fa/fa   0.66        -3.6 ± 2.0                                  citric acid                                                                   ______________________________________                                         .sup.a Fa/, lean rats;                                                        fa/fa, obese rats.                                                            .sup.b Each value is the mean ± S.E.                                       *P ≦ 0.05                                                         

EXAMPLE 19 Effect of (-)-threo-chlorocitric acid on body weight and foodintake in meal-fed Charles River rats (16 day study)

Two month old Charles River female rats (Charles River BreedingLaboratories, Wilmington, Mass.) were individually housed inwire-bottomed cages in a temperature-regulated (22° C.) light-controlledroom (12 hr light, 6 a.m. to 6 p.m. and 12 hr dark, 6 p.m. to 6 a.m.).Three groups of rats (9 to 10 rats per group) were meal-fed a singlemeal daily of G-70 diet from 8 to 11 a.m. for 5 days; on the sixth day,they were treated as follows: (1) control, (2) (-)-threo-chlorocitricacid (1.0 mmoles/kg, 227 mg/kg) administered by oral intubation 30 minbefore the 3 hr meal, and (3) (-)-threo-chlorocitric acid (1.1mmoles/kg, 250 mg/kg) as a dietary admixture for the 3 hr meal.Treatment was continued for 16 days. The amount of body weight gainedand food consumed during the study were determined.

    __________________________________________________________________________    RESULTS                                                                                              IBW.sup.a                                                                          FBW.sup.a                                                                           CBWG.sup.a                                                                          CFC.sup.a                                                                           Drug Ingested                   Treatment              g    g     g     g     mmoles (mg)/kg/day              __________________________________________________________________________    Control                197 ± 2.sup.b                                                                   219 ± 4                                                                          23 ± 2                                                                           211 ± 6                                                                          --                              (-)-threo-Chlorocitric acid (oral intubation)                                                        189 ± 3                                                                         163 ± 5***                                                                       -27 ± 4***                                                                       139 ± 6***                                                                       1.0 (227)                       (-)-threo-Chlorocitric acid (dietary admixture)                                                      192 ± 5                                                                         184 ± 8***                                                                       -8 ± 6***                                                                        157 ± 9***                                                                       1.1 (250)                       __________________________________________________________________________     .sup.a Abbreviations:                                                         IBW, initial body weight;                                                     FBW, final body weight;                                                       CBWG, cumulative body weight gain;                                            CFC, cumulative food consumption.                                             .sup.b Each value is the mean ± S.E.                                       ***P ≦ 0.001                                                      

EXAMPLE 20 Body consumption of meal-fed Charles River rats administered(-)-threo-chlorocitric acid (16 day study)

Two month old Charles River female rats (Charles River BreedingLaboratories, Wilmington, Mass.) were individually housed inwire-bottomed cages in a temperature-regulated (22° C.) light-controlledroom (12 hr light, 6 a.m. to 6 p.m. and 12 hr dark, 6 p.m. to 6 a.m.).Three groups of rats (9 to 10 rats per group) were meal-fed a singlemeal daily of G-70 diet from 8 to 11 a.m. for 5 days; on the sixth day,they were treated as follows: (1) control, (2) (-)-threo-chlorocitricacid (1.0 mmoles/kg, 227 mg/kg) administered by oral intubation 30 minbefore the 3 hr meal, and (3) (-)-threo-chlorocitric acid (1.1mmoles/kg, 250 mg/kg as a dietary admixture for the 3 hr meal. Treatmentwas continued for 16 days.

After 16 days of treatment, rats were killed by decapitation and bloodwas removed. Carcasses were weighed, saponified in alcoholic potassiumhydroxide, acidified, and extracted with petroleum ether according tothe method described by A. C. Sullivan, et al, Am. J. ClinicalNutrition, 30 767 (1977). The petroleum ether supernatants weretransferred to preweighed glass vials, evaporated immediately to drynessunder nitrogen, and reweighed. Total carcass lipid data are expressed ingrams and percentages of carcass weight. An aliquot of saponifiedcarcass extract was neutralized and total carcass nitrogen wasdetermined using the Kjeldahl procedure described in "Hawks'Physiological Chemistry," B. L. Oser, ed., McGraw Book Co., New York,N.Y., 1965, page 1214. Carcass protein data are expressed in grams andpercentages of carcass weight.

    __________________________________________________________________________    RESULTS                                                                                                     Carcass Lipid     Carcass Nitrogen                           Dose    Carcass Weight.sup.a                                                                            % of           % of                    Treatment    mmoles (mg)/kg                                                                        g        total (g)                                                                              carcass weight                                                                         total (g)                                                                           carcass                 __________________________________________________________________________                                                          weight                  Control      --      207 ± 4.sup.b                                                                       9.23 ± 0.79                                                                         4.44 ± 0.36                                                                         8.92 ± 0.32                                                                      4.31 ± 0.13          (-)-threo-Chlorocitric                                                                     1.0 (227)                                                                             155 ± 4***                                                                            2.74 ± 0.19***                                                                      1.94 ± 0.13***                                                                    6.57 ± 0.30                                                                      4.27 ± 0.22          acid (oral intubation)                                                        (-)-threo-Chlorocitric                                                                     1.1 (250)                                                                             176 ± 8**                                                                           7.01 ± 0.92                                                                         4.01 ± 0.45                                                                         7.16 ± 0.29                                                                      4.10 ± 0.14          acid (dietary admixture)                                                      __________________________________________________________________________     .sup.a Carcass includes body minus blood.                                     .sup.b Each value is the mean ± S.E.                                       **P ≦ 0.01                                                             ***P ≦ 0.001                                                      

EXAMPLE 21 Effect of (-)-threo-chlorocitric acid (oral administration)on body weight and food intake in meal-fed Charles River rats (17 daystudy)

Two month old Charles River female rats (Charles River BreedingLaboratories, Wilmington, Mass.) were individually housed inwire-bottomed cages in a temperature-regulated (22° C.) light-controlledroom (12 hr light, 6 a.m. to 6 p.m. and 12 hr dark, 6 p.m. to 6 a.m.).The animals (8 groups of 6 to 10 rats per group) were meal-fed a singlemeal daily of G-70 diet or chow from 8 a.m. to 11 a.m. for 7 days. Onthe 8th day, the animals were administered water or(-)-threo-chlorocitric acid at the indicated concentration by oralintubation 30 min before the 3 hr meal. Treatment was continued for 17days. Food cups were weighed immediately after the meal. The amount ofbody weight gained and food consumed during the period was determined.

    __________________________________________________________________________    RESULTS                                                                                         Dose     IBW.sup.a                                                                           FBW.sup.a                                                                           CBWG.sup.a                                                                           CFC.sup.a                       Treatment     Diet                                                                              mmoles (mg)/kg                                                                         g     g     g      g                               __________________________________________________________________________    Control       G-70                                                                              --        199 ± 6.sup.b                                                                   217 ± 5                                                                          18 ± 2                                                                            220 ± 7                      (-)-threo-Chlorocitric acid                                                                     0.25 (57)                                                                              198 ± 4                                                                          211 ± 7                                                                          12 ± 4                                                                            209 ± 10                                       0.50 (114)                                                                             195 ± 4                                                                          188 ± 7**                                                                        -6 ± 6**                                                                          176 ± 9***                                     1.00 (227)                                                                             198 ± 3                                                                          169 ± 6***                                                                       -29 ± 6***                                                                        153 ± 4***                   Control       Chow                                                                              --       199 ± 5                                                                          217 ± 6                                                                          19 ± 3                                                                            209 ± 5                      (-)-threo-Chlorocitric acid                                                                     0.25 (57)                                                                              214 ± 7                                                                          213 ± 7                                                                          -1 ± 4***                                                                         186 ± 9*                                       0.50 (114)                                                                             209 ± 5                                                                          177 ± 6***                                                                       -32 ± 6***                                                                        165 ± 9***                                     1.00 (227)                                                                             207 ± 3                                                                          176 ± 11**                                                                       -30 ± 7***                                                                        130 ± 13***                  __________________________________________________________________________     .sup.a Abbreviations:                                                         IBW, initial body weight;                                                     FBW, final body weight;                                                       CBWG, cumulative body weight gain;                                            CFC, cumulative food consumption.                                             .sup.b Each value is the mean ±  S.E.                                      *P ≦ 0.05                                                              **P ≦ 0.01                                                             ***P ≦ 0.001                                                      

EXAMPLE 22 Effect of (-)-threo-chlorocitric acid (oral administration)on total body fat in meal-fed Charles River rats (17 day study)

Two month old Charles River female rats (Charles River BreedingLaboratories, Wilmington, Mass.) were individually housed inwire-bottomed cages in a temperature-regulated (22° C.) light-controlledroom (12 hr light, 6 a.m. to 6 p.m. and 12 hr dark, 6 p.m. to 6 a.m.).The animals (8 groups of 6 to 10 rats per group) were meal-fed a singlemeal daily of G-70 diet or chow from 8 a.m. to 11 a.m. for 7 days. Onthe 8th day, the animals were administered water or(-)-threo-chlorocitric acid at the indicated concentration by oralintubation 30 min before the 3 hr meal. Treatment was continued for 17days. Food cups were weighed immediately after the meal.

After 17 days of treatment, rats were killed by decapitation and bloodwas removed. Carcasses and livers were weighed, saponified in alcoholicpotassium hydroxide, acidified, and extracted with petroleum etheraccording to the method described by A. C. Sullivan, et al, Am. J.Clinical Nutrition, 30 767 (1977). The petroleum ether supernatants weretransferred to preweighed glass vials, evaporated immediately to drynessunder nitrogen, and reweighed. Total carcass and lipid data areexpressed in grams and percentages of carcass weight.

    __________________________________________________________________________    RESULTS                                                                                                           Carcass Lipid.sup.a                                         Dose     Carcass Weight.sup.a                                                                             % of         % of               Treatment     Diet                                                                              mmoles (mg)/kg                                                                         g        total (g) control                                                                           % carcass                                                                              control            __________________________________________________________________________    Control       G-70                                                                                --       --     11.8 ± 1.97                                                                          100 5.43 ± 0.88                                                                         100                (-)-threo-Chlorocitric acid                                                                     0.25 (57)                                                                              196 ± 4**                                                                           8.98 ± 0.30                                                                          76  4.58 ± 0.09                                                                         84                                   0.50 (114)                                                                             177 ± 2***                                                                          7.85 ± 1.03                                                                          67  4.43 ± 0.56                                                                         82                                   1.00 (227)                                                                             169 ± 6***                                                                           5.95 ± 1.28*                                                                        50  3.48 ± 0.68                                                                         64                 Control       Chow                                                                                --     208 ± 3                                                                             9.56 ± 1.23                                                                          100 4.62 ± 0.63                                                                         100                (-)-threo-Chlorocitric acid                                                                     0.25 (57)                                                                              213 ± 4                                                                             9.64 ± 0.80                                                                          101 4.56 ± 0.42                                                                         99                                   0.50 (114)                                                                             173 ± 4***                                                                            4.80 ± 0.48**                                                                      50   2.70                                                                                  58-. 0.17*                           1.00 (227)                                                                             163 ± 7***                                                                            3.92 ± 0.37***                                                                     41    2.40                                                                                 52-. 0.22**        __________________________________________________________________________     .sup.a Carcass includes body minus 4 to 5 ml of blood.                        .sup.b Each value is the mean ± S.E.                                       *P ≦ 0.05                                                              **P ≦ 0.01                                                             ***P ≦ 0.001                                                      

EXAMPLE 23 Effect of stereoisomers of chlorocitric acids on lipidsynthesis in isolated rat hepatocytes Fatty Acid and CholesterolSynthesis and CO₂ Production in Isolated Hepatocytes

Female Charles River rats were fasted 48 hr, then meal-fed a 1% cornoil, 70% glucose diet for 7 to 14 days from 8 to 11 a.m. The isolatedrat hepatocytes were prepared by perfusing the liver in situ. Thehepatocytes were incubated in an oscillating water bath at 37° C. for 60min. Each flask contained a total of 2.1 ml volume, consisting of 1 mlisolated rat hepatocytes (10-20 mg dry weight cells), 1 mlKrebs-Henseleit bicarbonate buffer pH 7.4, 16.5 mM glucose, 1 μmoleL-alanine, 1μ Ci [U-¹⁴ C]alanine, 1 mCi ³ H₂ O, and 2 mM inhibitor in H₂O at pH 7.4. All incubations were done in triplicate and all experimentswere repeated at least twice. CO₂ was collected in each flask followingthe 60 min incubation by adding 0.3 ml ethanolamine:2-methoxyethanol(1:2) to the center well, 0.4 ml of 62.5% citric acid to the cell media,and incubating for 45 min. The contents of the center well weretransferred to scintillation counting fluid and ¹⁴ CO₂ content wasdetermined. Ther media was saponified, extracted with hexane,precipitated with digitonin, washed, and counted (to determine the rateof cholesterogenesis). The media was then acidified, extracted withhexane, and the extract counted (to determine the rate of lipogenesis).The conversion of ³ H₂ O and [¹⁴ C]alanine into fatty acids or sterolswas determined in a PDS/3, Mark II liquid scintillation counting system.Data were expressed as nmoles ³ H₂ O and [¹⁴ C]alanine converted intofatty acids or cholesterol, and nmoles [¹⁴ C]alanine oxidized to ¹⁴ CO₂per mg dry weight cells per 60 min.

    __________________________________________________________________________    RESULTS                                                                                     Fatty Acid Synthesis                                                                             Cholesterol Synthesis                                                                            CO.sub.2 Production                  Dose                                                                             .sup.3 H.sub.2 O converted                                                            [.sup.14 C]alanine converted                                                             .sup.3 H.sub.2 O converted                                                            [.sup.14 C]alanine                                                                       [.sup.14 C]alanine                                                            converted                 Compound   mM % of control                                                                          % of control                                                                             % of control                                                                          % of control                                                                             % of                      __________________________________________________________________________                                                        control                   Control    -- 100     100        100     100        100                       Trisodium (-)-threo-                                                                     2   54**    30**       60**     18***    103                       hydroxycitrate                                                                (+)-threo-Chlorocitric                                                                   2   93     102         60***    69***      114***                  acid                                                                          (-)-threo-Chlorocitric                                                                   2  106      124*      116     88*        83*                       acid                                                                          (-)-threo-Chlorocitric                                                                   2   95      96         98     92          91                       acid                                                                          (+)-threo-Chlorocitric                                                                   2   94      97        120      88**      100                       acid                                                                          __________________________________________________________________________     *P ≦ 0.05                                                              **P ≦ 0.01                                                             ***P ≦ 0.001                                                      

EXAMPLE 24 Effects of (±)-threo-chlorocitric acid compared to trisodium(-)-threo-hydroxycitrate on fatty acid synthesis in pair-feedingexperiments

Female rats of the Charles River CD strain (Charles River BreedingLaboratories, Wilmington, Mass.) weighing 120-160 g (ca. 16 week old)were housed individually in wire-bottomed cages in atemperature-regulated (22 C), light-controlled room (12 hour light 6A.M.-6 P.M., and dark 6 P.M.-6 A.M.). They had free access to water andwere fed a commercial diet (Purina Rodent Chow, Ralston Purina Co., St.Louis, Mo.) ad libitum for at least 1 week prior to the experiment.Animals were fasted 48 hours, then meal-fed a synthetic diet (G-70)daily from 8-11 A.M. for the remainder of the experiment. Foodconsumption and body weight were measured during the meal-feedingperiod. Body weights were randomized, so that each experimental grouphad an identical weight spread. Food spillage was measured daily.

Each group consisted of 8 to 10 (140 to 160 g) rats. Food intake wasmonitored for a 3 day period; the animals were dosed immediately beforethe 3 hr meal. Pair-fed animals were fed the amount of food which thedrug treated groups ate on the previous days (days 2 and 3).

Immediately after the 3 hour feeding period, rats were administeredintravenously a 0.25 ml saline (pH 7.4 to 7.6) solution with thefollowing composition: 12.3 mg alanine, 5μ Ci [¹⁴ C] alanine (specificactivity=156 mCi/mmole), 30.6 mg-ketoglutarate (as an amine acceptor fortransaminase) and 1 mCi [³ H] water (specific activity=100 mCi/g).Experiments indicated that [¹⁴ C] alanine was equivalent to either [¹⁴C] pyruvate or [¹⁴ C] lactate as a carbon precursor for lipogenesis. [³H] Water was employed to determine the total rate of lipogenesis, sincetritium is incorporated into fatty acids independent of the source ofcarbon precursors. Animals were killed by decapitation and bloodcollected in centrifuge tubes 30 minutes after the radioactive pulse,unless otherwise indicated. The specific radioactivity of the body waterof each rat was determined by counting a diluted serum aliquot in 10 mlfollowing cocktail: toluene (2.41), 2-methoxyethanol (1.61), naphthalene(320 g), and BBOT (2,5-bis-2-(5-tert-butylbenzoxazolyl)-thiophene, 16g). Liver, and small intestine was excised rapidly, weighed, andhomogenized in 15 ml H₂ O in a Virtis 45 Macro Homogenizer for 15 sec atca. 30,000 rpm. Duplicate 3 ml aliquots of whole homogenates weresaponified, extracted, and the absolute radioactivity (dpm) determined.Liver lipids were extracted totally or separated into fatty acids andcholesterol by anion exchange chromatography. It was determined by anionexchange chromatography that the total lipid extract of liver containedfatty acids (96-97%) and cholesterol (3-4%). Data are expressed asnmoles [¹⁴ C] alanine or μmoles [³ H] water converted into lipid/gtissue/30 minutes. The nmoles [¹⁴ C] alanine were calculated accordingto the injected load of alanine, as reported previously by A. C.Sullivan et al., Arch. Biochem. and Biophys., 150, 183 (1972). Theμmoles [³ H] water were determined as described previously by A. C.Sullivan et al., Lipids, 9, 121 (1974).

    __________________________________________________________________________    RESULTS                                                                                              Fatty Acid Synthesis                                                    Dose.sup.a                                                                          μmoles .sup.3 H.sub.2 O converted/                                                      nmoles [.sup.14 C]alanine converted/      Treatment        mmoles/kg                                                                           g liver/30 min                                                                             g liver/30 min                            __________________________________________________________________________    Pair-fed control --     21.3 ± 2.3.sup.b                                                                       714 ± 86                               Trisodium (-)-threo-hydroxycitrate                                                             2.63   14.4 ± 1.6*                                                                              279 ± 36**                           Pair-fed control --    25.6 ± 3.9                                                                              639 ± 69                               (±)-threo-Chlorocitric acid                                                                 1.32  21.4 ± 1.6                                                                              809 ± 67                               __________________________________________________________________________     .sup.a Doses were selected to produce a comparable reduction in food          intake.                                                                       .sup.b Each value is the mean ± S.E.                                       *P ≦ 0.05                                                              **P ≦ 0.01                                                        

EXAMPLE 25 Effect of chlorocitric stereoisomers on stomach emptying

Rats were trained to consume a 3 hr 70% glucose meal from 8 a.m. to 11a.m. daily. On the experimental day, compounds were administered orallyby intubation 30 min before a [¹⁴ C] glucose load was given also byintubation. The [¹⁴ C] glucose load consisted of 1.25μ Ci [U-¹⁴ C]glucose and 1.0 g glucose dissolved in water. Sixty minutes later therats were killed by decapitation; blood and stomachs were collected onice. Serum was analyzed for [¹⁴ C] radioactivity as follows: 0.5 ml ofserum was added to 10 ml of BBOT, an aqueous scintillation countingsystem (toluene [2.4 l], 2-methoxyethanol [1.6 l], naphthalene [320 g]and BBOT[2,5-bis-t-[5-tert-butylbenzoxazolylthiophine] [16 g], andradioactivity determined. Stomachs were cut longitudinally and washedexhaustively with saline. Total volume of stomach contents was measuredand 0.5 ml was added to 10 ml of BBOT, and radioactivity was determined.

Data are expressed as follows: dpm×10³ per ml of serum and dpm×10³ perstomach.

    __________________________________________________________________________    RESULTS                                                                                                   Stomach Contents                                                                       Serum                                                   Dose         dpm × 10.sup.3                                                               % of                                                                              dpm × 10.sup.3                                                                % of                               Treatment      mmoles (mg)/kg                                                                             total per stomach                                                                  control                                                                           total per ml                                                                        control                            __________________________________________________________________________    Control        --            884 ± 145.sup.a                                                                100 5.7 ± 0.3                                                                        100                                Trisodium Citrate                                                                            0.33 (97)    1180 ± 208                                                                      133 6.4 ± 0.1                                                                        112                                Trisodium (-)-hydroxycitrate                                                                 0.33 (101)   1973 ± 154***                                                                   223 4.5 ± 0.3*                                                                       79                                 (+)-threo-Chlorocitric acid                                                                  0.33 (75)    1946 ± 361*                                                                     220 3.4 ± 0.6*                                                                       60                                 (-)-threo-Chlorocitric acid                                                                  0.33 (75)    2760 ± 69***                                                                    312 1.9 ± 0.4***                                                                     33                                 (-)-erythro-Chlorocitric acid                                                                0.33 (75)    1491 ± 232                                                                      169 5.1 ± 0.4                                                                        90                                 (+)-erythro-Chlorocitric acid                                                                0.33 (75)    1832 ± 160**                                                                    207 5.0 ± 0.4                                                                        88                                 __________________________________________________________________________     .sup.a Each value is the mean ± S.E.                                       *P ≦ 0.05                                                              **P ≦ 0.01                                                             ***P ≦ 0.001                                                      

EXAMPLE 26 Effect of oral administration of (±)-threo-chlorocitric acid(+)-threo-chlorocitric acid and (-)-threo-chlorocitric acid on foodintake in dogs^(a)

Twelve beagle dogs of both sexes weighing 7.6-12.2 kg were used for thistest. The test compounds were contained in gelatin capsules for oraladministration to two dogs of each sex. The animals were weighed andfood consumption calculated daily excluding weekends throughout the testperiod. Each animal received 800 grams of diet daily consisting of 400grams of Wayne pellet dog food mixed with 400 ml distilled water. Theanimals received pyramiding oral doses of the test compound over thethree week period. On Monday and Tuesday of the first week the animalswere weighed and food consumption was recorded (control). The animalsreceived 3 mg/kg on Wednesday and 10 mg/kg on Friday of the first week.Doses of 30, 100 and 300 mg/kg were administered on Monday, Wednesdayand Friday respectively during the second week. A final dose of 1000mg/kg was administered on Monday of the third week.

    ______________________________________                                        RESULTS                                                                                Food Consumption                                                                    (±)-threo-                                                                            (+)-threo-                                                                             (-)-threo-                                                Chlorocitric                                                                             Chlorocitric                                                                           Chlorocitric                               Test Day                                                                             Dose    acid g     acid g   acid g                                     ______________________________________                                        -2, -1 --      479 ± 96.sup.c                                                                        610 ± 43                                                                            743 ± 54                                 1      3      616 ± 79                                                                              430 ± 109                                                                           450 ± 165*                               2     --      392 ± 140                                                                             502 ± 109                                                                           646 ± 83                                 3      10     460 ± 136                                                                             367 ± 114*                                                                          440 ± 121*                               6      30     511 ± 104                                                                             629 ± 64                                                                            406 ± 86**                               7     --      479 ± 116                                                                             397 ± 87*                                                                           668 ± 115                                8     100     299 ± 176                                                                             477 ± 75                                                                            142 ± 38***                              9     --      492 ± 110                                                                             585 ± 69                                                                            619 ± 62                                10     300     316 ± 109                                                                             436 ± 60                                                                             55 ± 39***                             13     1000    164 ± 61*                                                                             433 ± 125                                                                            56 ± 22***                             14     --      560 ± 136                                                                             518 ± 19                                                                            439 ± 163*                              15     --      543 ± 92                                                                              622 ± 69                                                                            583 ± 149                               16     --      624 ± 88                                                                              564 ± 106                                                                           600 ± 76                                17     --      645 ± 93                                                                              597 ± 36                                                                            677 ± 73                                20     --      800.sup.a  583 ± 17.sup.b                                                                      607 ± 193.sup.b                         21     --      561 ± 83                                                                              578 ± 79                                                                            644 ± 100                               22     --      649 ± 91                                                                              668 ± 57                                                                            800 ± 0                                 23     --      533 ± 118                                                                             516 ± 66                                                                            596 ± 121                               24 --  477 ± 114                                                                          539 ± 39                                                                              695 ± 79                                         27     --      518 ± 139                                                                             479 ± 92                                                                            732 ± 68                                28     --      663 ± 137                                                                             710 ± 91                                                                            775 ± 26                                ______________________________________                                         .sup.a One animal.                                                            .sup.b Two animals.                                                           .sup.c Each value is the mean ± S.E.                                       *P ≦ 0.05                                                              **P ≦ 0.01                                                             ***P ≦ 0.001                                                      

EXAMPLE 27 Effect of oral administration of (±)-threo-chlorocitric acid,(±)-threo-chlorocitric acid, and (-)-threo-chlorocitric acid on foodintake in dogs

Food intake was determined by the procedure at pyramiding dosesdescribed in EXAMPLE 26.

    ______________________________________                                        RESULTS                                                                                Food Consumption                                                                    (±)-threo-                                                                            (+)-threo-                                                                             (-)-threo-                                                Chloro-    Chloro-  Chloro-                                                   citric acid                                                                              citric acid                                                                            citric acid                                Test Day                                                                             Dose    % of control                                                                             % of control                                                                           % of control                               ______________________________________                                        -2, -1 --      100        100      100                                        1       3      129        70        60*                                       2      --       82        82       87                                         3       10      96         60*     59*                                        6       30     107        103        55***                                    7      --      100         65*     90                                         8      100      62        78         19***                                    9      --      103        96       83                                         10     300      66        71         7***                                     13     1000     34*       71         8***                                     14     --      117        85        59*                                       15     --      113        102      78                                         16     --      130        92       81                                         17     --      135        98       91                                         20     --       167.sup.a  96.sup.b                                                                               82.sup.b                                  21     --      117        95       87                                         22     --      135        110      108                                        23     --      111        85       80                                         24     --      100        88       94                                         27     --      108        79       99                                         28     --      138        116      104                                        ______________________________________                                         .sup.a One animal.                                                            .sup.b Two animals.                                                           *P ≦ 0.05                                                              **P ≦ 0.01                                                             ***P ≦ 0.001                                                      

EXAMPLE 28 Composition

The composition was prepared as follows:

    ______________________________________                                        Ingredients       Amount (mg/tablet)                                          ______________________________________                                        (-)-threo-Chlorocitric acid                                                                     50                                                          Polyvinylpyrrolidone                                                                            2                                                           Microcrystalline Cellulose                                                    (Avicil PH 101)   10                                                          Silicone Dioxide (Safoid 244)                                                                   1                                                           Magnesium Stearate                                                                              1                                                           ______________________________________                                    

(-)-threo-Chlorocitric acid was passed through a Fitzpatrick ComminutingMachine using a No. 1B plate, knives forward at medium speed. The milledcompound was granulated with a 40% (weight/weight) solution ofpolyvinylpyrrolidone in 2-propanol. The granulation was then passedthrough the Fitzpatrick machine using a No. 4 plate, knives forward atslow speed, and dried overnight at 55° C. to a granulation moisturecontent of equal to or less then 0.5%. The dried granulation was milledas before using No. 2A plates, knives forward at slow speed.Microcrystalline cellulose and silicone droxide was added to the drygranulation and mixed for ten minutes. Magnesium stearate was then addedand mixing was continued for an additional 5 minutes. Upon completion ofthe mixing, the formulation was compressed on a conventional tablettingmachine.

EXAMPLE 29 Mono-potassium (±)-threo-epoxyaconitate monohydrate

(a) Chlorination-Method.

A stock solution of potassium hydroxide and water was prepared by adding1320 g of potassium hydroxide pellets (ca. 85%) to 1000 ml of waterfollowed by the addition of 1500 g of ice. The solution was found tocontain 5.25 meq of potassium hydroxide per gram of solution and wasstored in a Nalgene container.

To 550 g (2.9 mol) of the stock solution was added 174 g (1.0 mol) oftrans aconitic acid portionwise over ca. 20 min, with cooling by theaddition of ice pellets (100 g). The pH of the resulting solution wasadjusted to a value of 7.2 by the dropwise addition of ca. 21 g (ca. 0.1mol) of the stock potassium hydroxide solution. Chlorine gas was addedto the reaction mixture cooled to about 5° to 7° by means of an externalacetone-dry ice bath, with stirring. After about 15 min, chlorine gaswas no longer absorbed by the reaction mixture. The flow of chlorine gaswas stopped, the reaction mixture was stirred for 10 to 15 min at about7° and the excess chlorine gas was purged using argon. The reactionmixture was cooled to about 20° and 135 g (about 2.05 mol) of potassiumhydroxide pellets (85%) were added in one portion with stirring. Afterstirring for about 5 min the resulting solution was cooled to 25° andthe pH of the reaction mixture was adjusted to a value of about 7.2 bythe dropwise addition of concentrated hydrochloric acid. 12 NHydrochloric acid (166 ml, 2.0 mol) was then added and the solution wasstored in a refrigerator at 0° to 5° overnight. The resultingprecipitate was collected on a filter and washed with cold ethanol-water(1:1) and ethanol. The filter cake was dried at 40° under house vacuumto give 245 g of a mixture of the mono-potassium salt of(±)-threo-epoxyaconitic acid as the monohydrate and potassium chloride.The solid material was dissolved in 400 ml of water at 60° and 300 ml ofethanol was added. The mixture was cooled to about 5° and the solidswere collected and washed with cold ethanol-water (1:1) and ethanol toyield 169 g of mono-potassium (±)-threo-epoxyaconitate monohydrate,having a neutralization equivalent of 123.6 g/equiv.

The product may be purified by recrystallization from water.

(b) Hypochlorination Method.

Chlorine gas was added to 419 g (2.2 mol) of the stock potassiumhydroxide solution and 75 ml of water cooled to 0°. The reaction mixturewas filtered to remove precipitated potassium chloride and the filtratewas added dropwise over about 15 min to a stirred solution of 174 g (1.0mol) of trans aconitic acid, 362 g (1.9 mol) of the stock potassiumhydroxide solution and 250 g of ice maintained at a reaction temperaturebetween -5° to 0°. The reaction mixture was stirred at 0° for about 15min and 7.0 g (0.1 mol) of chlorine gas was added by means of a gasdispersion tube as rapidly as possible. The reaction mixture was purgedof residual chlorine gas by means of argon, cooled to about 0° and 75 g(ca. 1.1 mol) of potassium hydroxide pellets (85%) were added in oneportion with stirring. The mixture was heated at 50° for about 10 min,cooled to about 25° and the pH of the solution was adjusted to a valueof about 7.2 by the dropwise addition of hydrochloric acid. Uponcompletion of the pH adjustment, 166 ml (2.0 mol) of 12 N hydrochloricacid was added with stirring and the solution was stored at 0° for 16hours. The precipitate was collected and washed with cold ethanol-water(1:1) and ethanol. The filter cake was dissolved in 350 ml of water andthe stirred solution was heated to 60°. Ethanol (ca. 300 ml) was addedand the mixture was stirred at 0° for 2 hours. The precipitate wascollected and washed with cold ethanol-water (1:1) and ethanol to give,after drying to constant weight, 158 g of the mono-potassium salt of(±)-threo-epoxyaconitic acid as the monohydrate having a neutralizationequivalent of 123.4 g/equiv.

(c) Alternative Hypochlorination Method.

To a solution of 116 g (0.67 mol) of trans aconitic acid, 370 g (ca.1.94 mol) of the stock potassium hydroxide solution (as prepared inmethod (b) and 250 g of ice, 11.4 g (ca. 0.06 mol) of the stockpotassium hydroxide solution was added dropwise to adjust the pH to thevalue of 7.2. To the resulting solution was added the stock potassiumhypochloride in one portion at about -10°. trans Aconitic acid (58.1 g,0.33 mol) was added in ca. 3 g portions to the stirred solution over ca.15 min so that the reaction temperature did not exceed -5°. After theaddition was complete, 8.3 ml (0.1 mol) of conc hydrochloric acid wasadded dropwise to the stirred solution. The reaction mixture was stirredat 0° for an additional 13 min and 7.0 g (ca. 0.1 mol) of chlorine gaswas added rapidly by means of a gas dispersion tube. After 5 min thereaction mixture was purged by argon gas and 75 g (ca. 1.1 mol) ofpotassium hydroxide pellets (85%) were added in one portion. Thereaction mixture was stirred at 50° for 15 min, cooled to roomtemperature and the pH was adjusted to a value of about 7.2 by thedropwise addition of concentrated hydrochloric acid. 12 N Hydrochloricacid (166 ml, 2.0 mol) was then added with stirring and the resultingsolution was stored at 5° overnight. The precipitate was collected andwashed with cold ethanol-water (1:1) and ethanol. The filter cake wasdissolved in 350 ml of water and diluted with 300 ml of ethanol. Thesolution was cooled to about 5° and after 2 hours the precipitate wascollected and washed with cold ethanol-water (1:1) and ethanol to give,after drying to constant weight, 161 g of mono-potassium salt of(±)-threo-epoxyaconitic acid having a neutralization equivalent of 123.6g/equiv.

(d) Peroxidation Method.

Sodium tungstate monohydrate (33.0 g, 0.1 mol) in ca. 75 ml of deionizedwater was passed through a prewashed column of 150 ml (0.3 equiv) ofAmberlite IR 120A ion-exchange resin (hydrogen ion form, ca. 2 equiv/ml)and the column was washed with 125 ml of deionized water. The combinedeluates were added to a solution of 348 g (2.0 mol) of trans aconiticacid and 800 ml of deionized water maintained at a temperature of 62°.Upon completion of the addition of the combined eluates, 250 ml (2.5mol) of 30% of hydrogen perioxide solution was added in one portion. Thereaction mixture was stirred for 30 min and an additional 75 ml of 30%of hydrogen peroxide solution was added in one portion at a reactiontemperature of about 65°. After 4 hours, the solution was cooled to 50°and 400 g (2.1 mol) of the stock potassium hydroxide solution was addedwith stirring. Ethanol (1.3 l) was added and the mixture was cooled to0° with stirring. After 3 hours the precipitate was collected and washedwith cold ethanol-water (1:1) and ethanol to give, after drying toconstant weight, 406 g of mono-potassium (±)-threo-epoxyaconitic acidmonohydrate.

EXAMPLE 30 (±)-threo-Epoxyaconitic acid.

Concentrated sulfuric acid (102.2 g, 1.0 mol) was added dropwise to asolution of 246 g (1.0 mol) of mono-potassium (±)-threo-epoxyaconiticacid, monohydrate, and 450 ml of acetone, with stirring at 20° to 23°.After the addition was complete, the reaction mixture was stirred atroom temperature for an additional 30 min. The precipitated potassiumhydrogen sulfate was collected and washed with acetone. The combinedfiltrate and washings were heated under reflux and 1.0 l of warm (ca.60°) 1,2-dichloroethane was added with stirring. The stirred solutionwas boiled and the volume was maintained at about 1.5 l by the periodicaddition of 1,2-dichloroethane, until the product began to crystallizefrom solution. The mixture was cooled to about 5° and, after stirringfor about 2 hours, the precipitate was collected and washed withacetone-1,2-dichloroethane (1:4) to give 151 g of(±)-threo-epoxyaconitic acid as the sesquihydrate.

The combined mother liquors were concentrated to a final volume of about1 l and the precipitate was collected and washed withacetone-1,2-dichloroethane (1:4) to give 45.8 g of the product as themonohydrate.

Concentration of the mother liquors and treatment of the precipitate ashereinbefore described afforded an additional 10.4 g of the monohydrate.

EXAMPLE 31 Resolution of (±)-threo-epoxyaconitic acid.

A solution of 38.0 g (0.2 mol) of (±)-threo-epoxyaconitic acid, 59.8 g(0.36 mol) of (+)-p-nitro-α-methylbenzylamine and 350 ml ofwater-methanol (1:49) was stirred at room temperature for 2 hours andstored at 0°-5° for 16 hours. The precipitate was collected, washed withwater-methanol (1:49) to afford, after drying under vacuum, 55.5 g ofcrude product. Recrystallization of the salt from water-methanol (1:49)gave 44.8 g of (+)-threo-epoxyaconitic acid, bis(+)-p-nitro-α-methylbenzylamine salt, having [α]₄₃₆ mm²⁵° C. +15.02° (c,2.0, water).

To a solution of 160 ml (0.181 mol) of 1.13 M hydrogen chloride andether, under an inert atmosphere, 44.4 g (0.082 mol) of theabove-obtained product was added portionwise with stirring at ca. 12°.The reaction mixture was allowed to warm to room temperature and wasstirred at room temperature for 30 min. The precipitate was collected,washed with ether and air dried to give 33 g of (+)-threo-epoxyaconiticacid.

The filtrates were combined and concentrated to dryness under reducedpressure. The residue was dissolved in hot ethyl acetate (about 70°) andabout 60 ml of carbon tetrachloride was added with stirring to the cloudpoint. The mixture was allowed to cool to room temperature and wasstored in a refrigerator overnight. The precipitate was collected,washed with ethyl acetate-carbon tetrachloride (1:3) and air dried togive an additional 16.4 g of product.

The (+)-threo-epoxyaconitic acid, monohydrate, so obtained had m.p.108°-110°, [α]₂₅ ^(D) +11.5° (c, 1.0 methanol) and neut equiv of 69.32g/equiv.

We claim:
 1. A process for the preparation of (±)-erythro-chlorocitric acid of the formula ##STR38## which comprises the steps of (a) contacting cis-aconitic acid of the formula ##STR39## or the anhydride thereof, cis-aconitic anhydride, of the formula ##STR40## with hydrogen peroxide in the presence of an alkali metal tungstate in an aqueous solvent containing about 2.5 molar equivalents of an alkali metal hydroxide to form a salt of (±)-erythro-epoxyaconitic acid of the formula ##STR41## wherein m is an alkali metal and R is hydrogen or M; and (b) contacting the salt of (±)-erythro-epoxyaconitic acid with an alkali metal chloride or an alkaline earth metal chloride in an aqueous solvent in the presence of about 3.5 molar equivalents of hydrochloric acid at a reaction temperature of about 50° C. to 80° C.
 2. The process of claim 1 wherein the alkali metal tungstate is sodium tungstate.
 3. The process of claim 1 wherein an alkali metal chloride is employed.
 4. The process of claim 3 wherein the alkali metal chloride is sodium chloride.
 5. The process of claim 4 wherein the reaction temperature of step (a) is about 20° C. to about 50° C.
 6. The process of claim 1 wherein the alkali metal hydroxide is sodium hydroxide.
 7. The process of claim 1 wherein the aqueous solvent is water.
 8. The process of claim 1 wherein the reaction temperature of step (b) is about 70° C. 